Regulation of Carbon Partitioning to Respiration during Dark Ammonium Assimilation by the Green Alga Selenastrum minutum

Abstract

The assimilation of NH4 causes a rapid increase in respiration to provided carbon skeletons for amino acid synthesis. In this study we propose a model for the regulation of carbon partitioning from starch to respiration and N assimilation in the green alga Selenastrum minutum. We provide evidence for both a cytosolic and plastidic fructose-1,6-bisphosphatase. The cytosolic form is inhibited by AMP and fructose-1,6-bisphosphate and the plastidic form is inhibited by phosphate. There is only one ATP dependent phosphofructokinase which, based on immunological cross reactivity, has been identified as being localized in the plastid. It is inhibited by phosphoenolpyruvate and activated by phosphate. No pyrophosphate dependent phosphofructokinase was found. The initiation of dark ammonium assimilation resulted in a transient increase in ADP which releases pyruvate kinase from adenylate control. This activation of pyruvate kinase causes a rapid 80% drop in phosphoenolpyruvate and a 2.7-fold increase in pyruvate. The pyruvate kinase mediated decrease in phosphoonolpyruvate correlates with the activation of the ATP dependent phosphofructokinase increasing carbon flow through the upper half of glycolysis. This increased the concentration of triosephosphate and provided substrate for pyruvate kinase. It is suggested that this increase in triosephosphate coupled with the glutamine synthetase mediated decline in glutamate, serves to maintain pyruvate kinase activation once ADP levels recover. The initiation of NH4 assimilation causes a transient 60% increase in fructose-2,6-bisphosphate. Given the sensitivity of the cytosolic fructose-1,6-bisphosphatase to this regulator, its increase would serve to inhibit cytosolic gluconeogenesis and direct the triosephosphate exported from the plastid down glycolysis to amino acid biosynthesis.Over the last decade there has been a significant increase in our knowledge of the control of carbon partitioning from starch to sucrose in photosynthetic tissue (34). In the proposed models, most of the carbon exported from the chloroplast is TP (see Table I for complete list of abbreviations). In the cytosol, Fru-2,6-P2 plays an important role in regulating the conversion of TP to sucrose through its effects on cytosolic It is well established that nitrogen exerts a major influence on respiratory carbon metabolism (1,9,15,16,31,(35)(36)(37)41). The assimilation of NH4' enhances starch breakdown and the flow of carbon into TCA cycle intermediates which are then used in amino acid biosynthesis. At present there is no model which provides an integrative picture of the mechanism by which NH4' assimilation regulates this carbon partitioning. In the present study we report changes in cellular metabolites during the dark assimilation of NH4' by the Nlimited green alga Selenastrum minutum. We also report preliminary data on the localization and regulation of PFK, PFP, and FBPase in this organism. Together, these results allow us to propose an integrative model for the control of carbon partitionin...

Anaerobic Metabolism in the N-Limited Green Alga Selenastrum minutum

“…7), a potent activator of S. minutum PK, thus reinforcing its activation. This hypothesized mechanism for activation of PK and PFK following a transition to anaerobiosis is similar to that described for activation of glycolysis to provide carbon skeletons for NH4' assimilation (38).…”

“…The physiological concentrations of metabolite effectors of PK and PFK have been shown to be in the range to effect enzyme activity (38). Also, recent work has suggested that the in vivo activity of S. minutum PK may be limited by the availability of ADP (38). Based on this information and on examination of short-term metabolite changes following a transition to anaerobiosis, we are able to hypothesize the following mechanism for the activation of PK and PFK.…”

“…The kinetic and regulatory properties ofthe S. minutum cytosolic and plastidic PKs and the single PFK (plastidic) have been examined in detail (5,23). The physiological concentrations of metabolite effectors of PK and PFK have been shown to be in the range to effect enzyme activity (38). Also, recent work has suggested that the in vivo activity of S. minutum PK may be limited by the availability of ADP (38).…”

“…A second possibility is that by stimulating PFP, the forward (glycolytic) reaction may provide FBP for glycolysis or conversely, the reverse (gluconeogenic) reaction may provide PPi for use in sucrose mobilization (1,2,33,34). In the green alga Selenastrum minutum, there is no PFP (5,38), but a cytosolic FBPase does exist and is strongly inhibited by Fru-2,6-P2 (6,38). The absence of PFP in this alga means that a role of Fru-2,6-P2 in regulating cytosolic FBPase during changes in glycolytic flux can be tested unambiguously.…”

In Organello and in Vivo Evidence of the Importance of the Regulatory Sulfhydryl/Disulfide System and Pyruvate for Alternative Oxidase Activity in Tobacco

“…FCCP treatment approximately doubled the respiration rate (O 2 consumption) of the cells (data not shown), indicating that it had been restricted by the availability of ADP (Dry et al, 1987). The increased availability of ADP after FCCP addition might be expected to decrease the reduction state of the mitochondrial pyridine nucleotide pool (Neuburger et al, 1984;Day et al, 1987) and result in the activation of pyruvate kinase, causing an increase in pyruvate (Turpin et al, 1990;Plaxton, 1996). While the more oxidized pyridine nucleotide pool expected in the presence of FCCP would not favor AOX reduction (Vanlerberghe et al, 1995), the expected increase in pyruvate would favor the reduced form and may explain why FCCP brought about no significant change in the AOX protein form (Fig.…”