C3-C4 intermediacy in grasses: organelle enrichment and distribution, glycine decarboxylase expression, and the rise of C2 photosynthesis

C-2 photosynthesis in grasses is facilitated by organelle enrichment in tandem with enhanced levels of GDC in the carbon-concentrating cells consistent with changes in expression of a single GLDP gene.Photorespiratory glycine shuttling and decarboxylation in bundle sheath (BS) cells exhibited by C-2 species is proposed to be the evolutionary bridge to C-4 photosynthesis in eudicots. To evaluate this in grasses, we compare anatomy, cellular localization of glycine decarboxylase (GDC), and photosynthetic physiology of a suspected C-2 grass, Homolepis aturensis, with these traits in known C-2 grasses, Neurachne minor and Steinchisma hians, and C-3 S. laxum that is sister to S. hians. We also use publicly available genome and RNA-sequencing data to examine the evolution of GDC subunits and enhance our understanding of the evolution of BS-specific GDC expression in C-2 and C-4 grasses. Our results confirm the identity of H. aturensis as a C-2 species; GDC is confined predominantly to the organelle-enriched BS cells in H. aturensis and S. hians and to mestome sheath cells of N. minor. Phylogenetic analyses and data obtained from immunodetection of the P-subunit of GDC are consistent with the hypothesis that the BS dominant levels of GDC in C-2 and C-4 species are due to changes in expression of a single GLDP gene in M and BS cells. All BS mitochondria and peroxisomes and most chloroplasts in H. aturensis and S. hians are situated centripetally in a pattern identical to C-2 eudicots. In S. laxum, which has C-3-like gas exchange patterns, mitochondria and peroxisomes are positioned centripetally as they are in S. hians. This subcellular phenotype, also present in eudicots, is posited to initiate a facilitation cascade leading to C-2 and C-4 photosynthesis.