Loss of the chloroplast transit peptide from an ancestral C₃ carbonic anhydrase is associated with C₄ evolution in the grass genus Neurachne

Neurachne is the only known grass lineage containing closely related C₃, C₃-C₄intermediate, and C₄species, making it an ideal taxon with which to study the evolution of C₄photosynthesis in the grasses. To begin dissecting the molecular changes that led to the evolution of C₄photosynthesis in this group, the complementary DNAs encoding four distinct β-carbonic anhydrase (CA) isoforms were characterized from leaf tissue of Neurachne munroi (C4), Neurachne minor (C₃-C₄), and Neurachne alopecuroidea (C₃). Two genes (CA1 and CA2) each encode two different isoforms: CA1a/CA1b and CA2a/CA2b. Transcript analyses found that CA1 messenger RNAs were significantly more abundant than transcripts from the CA2 gene in the leaves of each species examined, constituting ∼99% of all β-CA transcripts measured. Localization experiments using green fluorescent protein fusion constructs showed that, while CA1b is a cytosolic CA in all three species, the CA1a proteins are differentially localized. The N. alopecuroidea and N. minor CA1a isoforms were imported into chloroplasts of Nicotiana benthamiana leaf cells, whereas N. munroi CA1a localized to the cytosol. Sequence analysis indicated an 11-amino acid deletion in the amino terminus of N. munroi CA1a relative to the C₃and C₃-C₄proteins, suggesting that chloroplast targeting of CA1a is the ancestral state and that loss of a functional chloroplast transit peptide in N. munroi CA1a is associated with the evolution of C₄photosynthesis in Neurachne spp. Remarkably, this mechanism is homoplastic with the evolution of the C₄-associated CA in the dicotyledonous genus Flaveria, although the actual mutations in the two lineages differ.