Does C4 Photosynthesis Occur in Wheat Seeds?

Our recent report of new evidence for C4 photosynthesis in wheat seeds (Rangan et al., 2016) has been met with skepticism (Busch and Farquhar, 2016), maybe because this has been such a long-running controversy. Much of the confusion has been because early work did not distinguish photosynthesis in the pericarp of the seed (Fig. 1) from that in the covering glumes. Glumes, like leaves in wheat, capture CO2 from the atmosphere by C3 photosynthesis. However, RNA-Seq analysis demonstrates the expression of a complete C4 pathway in the seed with a C4-specific form of PEP carboxylase expressed specifically in the pericarp and aleurone capturing carbon released by respiration in the endosperm. Decarboxylation by a C4-specific malic enzyme in the inner pericarp supplies CO2 to a C3 pathway. The decarboxylation of malate specifically in the pericarp is suggested by comparison of the levels of C4-specific malic enzyme expression in the pericarp with the low levels in the endosperm. This provides a source of concentrated carbon dioxide for Rubisco in the pericarp, acting like the C4 pathway in leaves that effectively concentrates CO2 at the bundle sheath chloroplast for fixation in a C3 process. Unlike the situation in wheat leaves, high concentrations of CO2 in the endosperm cannot be fixed through photosynthesis due to the lack of light penetration, but capture as malate allows processing to generate higher CO2 concentrations for the Rubisco expressed very specifically in the photosynthetic pericarp. Analysis of the genome shows specific genes encoding the C4 enzymes that are distinct from those encoding the C3 pathway. These genes are expressed in a highly tissue-specific way in the pericarp and endosperm. Analysis of the specificity of wheat seed enzymes supports this interpretation. Labeling studies using C-labeled CO2 provide overwhelming support for the capture of carbon as malate in these tissues and the flow of this carbon into C3 photosynthesis (Nutbeam andDuffus, 1976; Singal et al., 1986). Confusion on this issue has been deepened by a labeling study (Bort et al., 1995) that did not recognize the difference between glumes capturing external CO2 and the inner seed capturing respired carbon. This study claimed evidence against C4 photosynthesis based upon feeding CO2 to the intact ear rather than respired carbon diffusing outwards from the endosperm despite very early work establishing the later as the source of carbon. Further misinterpretation may have resulted from an expectation that seed photosynthesiswould use free CO2 as a substrate. The endosperm of the developing seed maintains a high pH with a drop in pH only happening during germination. The native substrate in the developing seed tissues would be bicarbonate acting as a substrate for the large amounts of C4-specific PEP carboxylase found in the aleurone and pericarp. We therefore propose that there is no credible evidence opposing these reports of C4 activity in wheat. Study of tissues outside the pericarp and a lack of recognition by some of respiration in the endosperm as the source of carbon have confused the issue.