C4 photosynthesis is the product of several biochemical and structural adaptations that concentrate CO2 at the site of Rubisco (for ribulose-1,5-bis-phosphate carboxylase/oxygenase), thereby increasing photosynthetic efficiency in conditions that promote photorespiration. Having evolved independently up to 66 times in angiosperms, the C4 pathway represents a striking example of convergent evolution (Sage et al., 2012). Typically, C4 photosynthesis depends on the metabolic interaction between two cell types. CO2 is initially fixed as a four-carbon compound in mesophyll cells and is then concentrated in bundle sheath cells, which contain Rubisco and other enzymes of the Calvin cycle (Hatch et al., 1975). The glycine decarboxylase complex (GDC), which catalyzes the release of CO2 from the metabolites of photorespiration, is largely restricted to bundle sheath cells in C3-C4 intermediate and C4 species, where it functions as a CO2 pump that further concentrates CO2 in the vicinity of Rubisco. Given that the GDC is expressed throughout photosynthetic tissues in C3 plants and is restricted to bundle sheath cells in C3-C4 and C4 species, this spatial confinement of GDC already in C3-C4 intermediates is thought to represent an important step in the evolution of C4 photosynthesis (Sage, 2004; Sage et al., 2012).
The GDC consists of four proteins (P, L, T, and H), with the P protein (encoded by GLDP) constituting the decarboxylase (Oliver and Raman, 1995). Schulze et al. (pages 2522–2535) investigated how the bundle sheath–specific expression of GDC was established during the evolution of C4 photosynthesis in the genus Flaveria (yellowtops), which includes C3, C4, and C3-C4 intermediate species. First, they demonstrated that the GLDP genes in Flaveria clustered into three groups (A, B, and C) and that only two of these (A and B) accumulated in leaves and were involved in photorespiration. Flaveria trinervia, a C4 species, contained a functional A-cluster gene (GLDPA) and a B-cluster pseudogene (GLDPB*), whereas the C3 species examined contained functional genes in both of these clusters. The authors then fused the 5′-flanking sequences of the GLDPA genes from two of the C3 species to β-glucuronidase and expressed these fusions in Flaveria bidentis, a C4 species. Both of these genes were strongly expressed in the bundle sheath cells but not in mesophyll cells (see figure). Thus, the last common ancestor of extant C3 and C4 Flaveria spp appears to have had a bundle sheath–specific GLDP gene. A similar analysis demonstrated that the group B GLDP gene of a C3 Flaveria species was expressed in all photosynthetic tissues of a transgenic C4 species.
Group A and B GLDP genes exhibit different expression patterns in leaves. β-glucuronidase (GUS) reporter analysis revealed that expression of a GLDPA gene from a C3 Flaveria species was restricted to the bundle sheath cells when transgenically expressed in a C4 species (left), whereas a GLDPB gene was ubiquitously expressed (right). Images show GUS activity in leaf sections of transgenic C4 plants harboring GUS reporter constructs. Scale bar = 50 μm. (Adapted from Schulze et al. [2013], Figures 2A and 3A.)
Interestingly, RNA-seq analysis showed that C3-C4 intermediate species had lower levels of group B GLDP transcripts than did a C3 reference and that most C4 species examined had none. In contrast, levels of group A GLDP transcripts were most abundant in C3-C4 intermediates and low in C4 species.
Based on these results, the authors present a model that explains how GLDP expression gradually became specific to the bundle sheath cells in Flaveria. The C3 species contain two versions of the GLDP gene, one of which is expressed exclusively in bundle sheath cells, and the other of which is expressed ubiquitously in leaves. During the evolution of C4 species, the ubiquitously expressed version became a pseudogene, leaving only the bundle cell-specific version of GLDP.
References
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