Characterization of C 3 –C 4 intermediate species in the genus Heliotropium L. (Boraginaceae): anatomy, ultrastructure and enzyme activity

Photosynthetic pathway characteristics were studied in nine species of Heliotropium ( sensu lato , including Euploca ), using assessments of leaf anatomy and ultrastructure, activities of PEP carboxylase and C 4 acid decarboxylases, and immunolocalization of ribulose 1·5‐bisphosphate carboxylase/oxygenase (Rubisco) and the P‐subunit of glycine decarboxylase (GDC). Heliotropium europaeum , Heliotropium calcicola and Heliotropium tenellum are C 3 plants, while Heliotropium texanum and Heliotropium polyphyllum are C 4 species. Heliotropium procumbens and Heliotropium karwinskyi are functionally C 3 , but exhibit ‘proto‐Kranz’ anatomy where bundle sheath (BS) cells are enlarged and mitochondria primarily occur along the centripetal (inner) wall of the BS cells; GDC is present throughout the leaf. Heliotropium convolvulaceum and Heliotropium greggii are C 3 –C 4 intermediates, with Kranz‐like enlargement of the BS cells, localization of mitochondria along the inner BS wall and a loss of GDC in the mesophyll (M) tissue. These C 3 –C 4 species of Heliotropium probably shuttle photorespiratory glycine from the M to the BS tissue for decarboxylation. Heliotropium represents an important new model for studying C 4 evolution. Where existing models such as Flaveria emphasize diversification of C 3 –C 4 intermediates, Heliotropium has numerous C 3 species expressing proto‐Kranz traits that could represent a critical initial phase in the evolutionary origin of C 4 photosynthesis.