Directed Mutation of the Rubisco Large Subunit of Tobacco Influences Photorespiration and Growth

Abstract

The gene for the large subunit of Rubisco was specifically mutated by transforming the chloroplast genome of tobacco (Nicotiana tabacum). Codon 335 was altered to encode valine instead of leucine. The resulting mutant plants could not grow without atmospheric CO 2 enrichment. In 0.3% (v/v) CO 2 , the mutant and wildtype plants produced similar amounts of Rubisco but the extent of carbamylation was nearly twice as great in the mutants. The mutant enzyme's substrate-saturated CO 2 -fixing rate and its ability to distinguish between CO 2 and O 2 as substrates were both reduced to 25% of the wild type's values. Estimates of these parameters obtained from kinetic assays with the purified mutant enzyme were the same as those inferred from measurements of photosynthetic gas exchange with leaves of mutant plants. The Michaelis constants for CO 2 , O 2 , and ribulose-1,5-bisphosphate were reduced and the mutation enhanced oxygenase activity at limiting O 2 concentrations. Consistent with the reduced CO 2 fixation rate at saturating CO 2 , the mutant plants grew slower than the wild type but they eventually flowered and reproduced apparently normally. The mutation and its associated phenotype were inherited maternally. The chloroplast-transformation strategy surmounts previous obstacles to mutagenesis of higher-plant Rubisco and allows the consequences for leaf photosynthesis to be assessed.

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

“…This was initially evident from the approximately 2-fold higher carbamylation status of the hybrid Rubisco. As seen in transplastomic tobacco lines producing 50% less Rubisco or a kinetically inferior mutant (Whitney et al, 1999), the high carbamylation status presumably compensates for the lower carboxylation potential. Light transient gas-exchange analyses also showed the activation constant for L s S t was 25% faster than wild-type tobacco Rubisco, indicating the hybrid enzyme was adequately regulated by tobacco Rubisco activase (Fig.…”

“…Intriguingly in subsequent RNA blots using fully denaturing formaldehyde gels we find the rbcL S mRNA is smaller than the native rbcL mRNA, indicating the mature 3# UTR of rbcL extends further downstream (data not shown). In support of this, insertion of a promoterless aadA gene a further 91-bp 3# to that in tobacco Rst does not perturb rbcL abundance or Rubisco production and still enables transcription of an additional, less abundant (approximately 20% of the rbcL mRNA) rbcL-aadA transcript (Whitney et al, 1999;Whitney and Andrews, 2003). Clearly, the 3# UTR does have a pervasive influence on the steady-state levels of chloroplast mRNAs and to what extent lengthening the 3# UTR would improve rbcL S transcript abundance, and reduce transcriptional read through of the rbcL S -aadA message, remains to be examined.…”

“…V c max measurements in bold italics are those made using leaf tissue from tissue culture grown plants; see Supplemental Figure S3 for details. Superscript a indicates data from Whitney et al (1999) and superscript b indicates data from Kanevski et al (1999).…”

“…Limitations in the catalytic properties and content of L s S t were proposed to perturb autotrophic growth in air of tobacco Rst plants grown in soil. Analogous to other tobacco transgenic lines expressing less Rubisco (Rodermel et al, 1988;Hudson et al, 1992) or catalytically perturbed foreign or mutated Rubiscos (Whitney et al, 1999;Whitney and Andrews, 2001b), the T 1 generation of the tobacco Rst line Nt-pIK83-1 (Kanevski et al, 1999) could be grown in soil in air enriched with CO 2 . The phenotype of the T 1 progeny was different to wild-type tobacco (see figure 2 in Andrews and Whitney, 2003) and to ensure the phenotype was not a consequence of unwanted nuclear mutations the tobacco Rst flowers were repeatedly backcrossed with wild-type pollen.…”

Improved temperature response functions for models of Rubisco-limited photosynthesis

“…The temperature dependence of V o,max has been assumed to be a constant proportion of V c,max over a range of temperature at 0·21 ¥ V c,max (Farquhar et al 1980;Farquhar & von Caemmerer 1982) though numerous values ranging from 0·19 to 0·77 ¥ V c,max have been determined (Badger & Andrews 1974;Badger & Collatz 1977;Jordon & Ogren 1981, 1984Makino, Mae & Ohira 1988;Whitney et al 1999;von Caemmerer 2000). Although numerous studies report values for the ratio of V o,max /V c,max at 25°C, very few studies have determined the temperature response of this ratio.…”