Characterization of C3-C4 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.