Ancestral Reconstruction and C3 Bundle Sheath Transcript Abundance in the Paniceae Grasses Indicate the Foundations for all Three Biochemical C4 Sub-Types Were Likely Present in the Most Recent Ancestor

In C4 plants the enzymatic machinery underpinning photosynthesis can vary, with for example, three distinct C4 acid decarboxylases being used to release CO2 in the vicinity of RuBisCO. For decades, these decarboxylases have been used to classify C4 species into three biochemical sub-types. However, more recently the notion that C4 species mix and match C4 acid decarboxylases has increased in popularity and, as a consequence, the validity of specific biochemical sub-types has been questioned. Using species from the grass tribe Paniceae we show that whilst transcripts encoding multiple C4 acid decarboxylases accumulate in bundle sheath cells in some species, in others, transcripts encoding only one enzyme are detected. In addition, a method that allows isolation of bundle sheath cells from a C3 species within the Paniceae, Sacciolepis indica, was developed. Deep sequencing of bundle sheath preparations from all four species combined with ancestral state reconstruction support the notion that the three biochemical C4 sub-types found in the Paniceae existed together in their most recent common ancestor. Thus, these species likely inherited the functional building blocks of all three C4 pathways. We conclude that classification of C4 plants into the classical biochemical sub-types is still appropriate for some species, and that evolution of this trait has been facilitated by characteristics of the ancestral C3 bundle sheath and made use of multiple convergent routes involving either one or multiple C4 acid decarboxylases.