4.3 On the source of carbons for glutamate synthesis in illuminated guard cells
The connection between the TCA cycle and Glu metabolism is well-established (Araújo et al. 2012, 2013; O’Leary & Plaxton 2020). 13C-NMR studies indicate that the synthesis of Glu in illuminated leaves strongly depends on stored compounds (Tcherkezet al. 2009; Gauthier et al. 2010; Abadie et al.2017), which are presumably accumulated in the previous night period (Cheung et al. 2014). This is likely an alternative to overcome the light-inhibition of PDH (Tovar-Méndez et al. 2003; Zhanget al. 2021), an important source of carbons for the TCA cycle and associated pathways (Reid et al. 1977). The degradation of labelled pyruvate by PDH or PC would provide two and three labelled carbons into acetyl-CoA and OAA, respectively. Once synthesized, the acetyl-CoA could be used for citrate synthesis or exported from the mitochondria for fatty acid synthesis (Lonien & Schwender 2009). However, export of citrate for fatty acid synthesis is unlikely to hold true given that light exposure triggers fatty acid degradation in guard cells (McLachlan et al. 2016). It seems likely that fully labelled OAA is synthesized by the activity of both PEPc and PC, which is in turn simultaneously used to the synthesis of succinate through the activity of MDH and fumarase through the C4-branch of the TCA cycle and the synthesis of Glu through the C6(5)-branch of the TCA cycle. In parallel, PDH-derived acetyl-CoA would also contribute to the labelling of Glu through the C6(5)-branch of the TCA cycle.
This labelling pattern throughout the TCA cycle and associated pathways consider the incorporation of non-labelled carbons into malate and citrate. Alternatively, succinate, that is strongly labelled in the light, but not in the dark, could be used as a substrate for Glu synthesis. In this scenario, the TCA cycle would occur in a counterclockwise direction between succinate and 2-OG in illuminated guard cells. Although this possibility has never been confirmed in plants, reverse TCA cycle flux modes have been identified in certain bacteria, being a route to assimilate CO2 through the activities of 2-oxoglutarate dehydrogenase (OGDH) and isocitrate dehydrogenase (IDH) in the counterclockwise direction (Evans et al. 1966; Buchanan & Arnon 1990; Mall et al. 2018; Steffenset al. 2021). Thus, beyond providing carbon skeletons for Glu synthesis, a reverse mode of operation of the TCA cycle would also contribute to increase the CO2 assimilated in guard cells. However, this hypothesis is unlikely to occur given the biochemical characteristics of OGDH, that is improbable to occur in the reverse (counterclockwise) direction (Araújo et al. 2013), and the fact that this would depend on the activity of 2-OG synthase (E.C 1.2.7.3), that has only been described in bacteria’s (Buchanan & Evans 1965; Buchanan & Arnon 1990; Yoon et al. 1996, 1997; Dörner & Boll 2002; Yamamoto et al. 2010). Therefore, our results collectively suggest that the carbon derived from PEPc-mediated CO2 assimilation is simultaneously used to support gluconeogenesis, the TCA cycle and Glu synthesis and that previously stored citrate and malate may also be an important source of carbons for the TCA cycle and associated pathways.