Anaerobic Metabolism in the N-Limited Green Alga Selenastrum minutum

Abstract

The onset of anaerobiosis in darkened, N-limited cells of the green alga Selenastrum minutum (Naeg.) Collins elicited the following metabolic responses. There was a rapid decrease in energy charge from 0.85 to a stable lower value of 0.6 accompanied by rapid increases in pyruvate/phosphoenolpyruvate and fructose-1,6-bisphosphate/fructose-6-phosphate ratios indicating activation of pyruvate kinase and 6-phosphofructokinase, respectively. There was also a large increase in fructose-2,6-bisphosphate, which, since this alga lacks pyrophosphate dependent 6-phosphofructokinase, can be inferred to inhibit gluconeogenic fructose-1,6-bisphosphatase activity. flux during anoxia (the Pasteur effect) is accompanied by large increases in FBP/F6P and pyr/PEP ratios (37). A Pasteur effect also has been shown to occur in green algae (13, 28) but these observations have not been accompanied by a detailed analysis of changes in glycolytic intermediates. There has been speculation that PFK is also important in regulating anaerobic carbon metabolism in green algae (13).Fru-2,6-P2 is an important effector ofplant PFP and FBPase and therefore a key regulator of plant respiration (2). Studies have reported increases in Fru-2,6-P2 in response to anoxia in higher plants (22,27) yet it is unclear how Fru-2,6-P2 contributes to increased glycolytic flux under these conditions. One possibility is that by inhibiting the cytosolic FBPase it stops gluconeogenic carbon flow. 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 higher plants, anaerobiosis has been shown to lead to an accumulation of succinate (9,25,26), and Ala (9,20,25,26,30,36) and a decline in Asp (9,36

Anaerobic Metabolism in the N-Limited Green Alga Selenastrum minutum

“…This is supported, in part, by the operation of a partial reductive TCA cycle from OAA to succinate (24). This pathway results in accumulation of succinate as a fermentative end-product contributing to redox balance and energy metabolism under anoxia (5,25).…”

“…Other studies have dealt with the accumulation of N containing compounds under anoxia. It has been observed that there are large fluxes of 14C to Ala from '4C-sugars in anoxic plant tissue (7,17,18) and that Ala accumulates in anoxic plant tissue (8,12,13,17,20,25). It anoxia (6).…”

Purification and Characterization of an Anaerobically Induced Alanine Aminotransferase from Barley Roots

“…Alanine has the advantage that it is a biocompatible solute and is retained in the cell (9) (25). In this scheme, the amino group of aspartate is conserved by transaminating pyruvate to alanine.…”

Normal Growth of Transgenic Tobacco Plants in the Absence of Cytosolic Pyruvate Kinase

“…After a stable rate was obtained (about 10 min), the lights were turned out and respiratory 02 consumption was monitored until a stable rate was recorded (about 10 min). Metabolite levels were determined from dark-adapted leaf discs (15 min) quenching in liquid N2 as previously described (31).…”

“…PKc cDNA (1) fused to a pea Rubisco small subunit transit peptide (RBCS-SSTP) was used to replace the GUS gene of the binary transformation vector pBI121 (13). NPT (1) was joined in-frame at the first methionine codon behind a fragment containing 31 (6), 2% (w/v) sucrose, and 0.8% (w/v) Difco Bacto agar. Leaf explants were excised and precultured for 2 d on N. tabacum cell suspension nurse cultures using the double filter paper technique (11) on MSB5 medium with 1 mg/mL 6-benzylaminopurine, 0.1 mg/mL a-naphthalene acetic acid, 3% (w/v) sucrose, and 0.6% (w/v) agarose (FMC, Seakem, ME).…”