Fig. 1.

Flaveria rubisco L-subunit sequence and expression in tobacco chloroplasts. (A) Comparison of Flaveria rbcL sequences that differ only in L-subunit substitutions at amino acids 149, 265, and 309 (20). (B) The transforming plasmid pLEV4 contains a homologous plastome flanking sequence (indicated by dashed lines; numbering indicates region of sequence integration relative to plastome sequence; GenBank ID Z00444) that directed integration of the rbcL transgenes and a promoterless aadA-selectable marker gene into the plastome of the tobacco master line, ^cmtrL (23). The L-subunit amino acid differences at residues 149 (white triangle), 265 (black triangle), and 309 (gray triangle) in the tob^LEV4 (tobacco rbcL control), tob^pring (F. pringlei rbcL), tob^flo (F. floridana rbcL), tob^bid (F. bidentis rbcL), tob^flo-bid (chimeric F. floridana–F. bidentis rbcL), tob^bid-flo (chimeric F. bidentis–F. floridana rbcL), and tob^149A,265I (mutated tobacco rbcL) transplastomic tobacco (tob) lines are shown. Annealing locations of primers LSH, LSE, and the 5′ ΔrbcL probe (24) are shown. N, NheI; S, SalI cloning sites. (C) Nondenaturing PAGE analysis of soluble protein from comparable leaves of independent T₀ transplastomic lines, ^cmtrL, and wild-type tobacco (protein from 1.5 mm² of leaf was loaded per lane). Homoplasmic transformants produce only L₈S₈ rubisco (∼520 kDa) and not the ∼100-kDa R. rubrum L₂ rubisco produced in ^cmtrL (23). (D) Detection of rbcL and rbcL-aadA mRNA in total RNA from 3 mm² (for wild type) or 6 mm² (other samples) of the leaves sampled in C.