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.