Distinct C₄ sub-types and C₃ bundle sheath isolation in the Paniceae grasses

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 five species from the grass tribe Paniceae, we show that, although in some species transcripts and enzymes involved in multiple C₄ acid decarboxylases accumulate, in others, transcript abundance and enzyme activity is almost entirely from one decarboxylase. In addition, the development of a bundle sheath isolation procedure for a close C₃ species in the Paniceae enables the preliminary exploration of C₄ sub-type evolution.