Glycolytic Flux and Hexokinase Activities in Anoxic Maize Root Tips Acclimated by Hypoxic Pretreatment

Abstract

Severa1 enzyme activities were measured in extracts from acclimated and nonacclimated maize (Zea mays) root tips at pH 6.5 and 7.5, corresponding to cytoplasmic pH in anaerobiosis or aerobiosis, respectively, to determine what causes the decline of the glycolytic flux observed in anoxia in nonacclimated tips. We found that phosphorylation of hexoses by kinases was a major limiting step of glycolysis in anoxia. When fructose was substituted for glucose, glycolysis was slightly enhanced and survival improved, but neither matched that of acclimated tips. Decrease of kinase activities was not the result of proteolytic degradation but was more likely the result of inhibition by interna1 factors (low pH and low ATP). There was no evidence of induction during the hypoxic pretreatment of isoenzymes better adapted to the anoxic cellular environment. A preacclimation in hypoxia allows excised maize (Zea nzays) root tips supplemented with externa1 Glc to survive severa1 days in strict anoxia instead of less than 10 h in the absence of pretreatment Xia and Saglio, 1992). However, in the absence of exogenous Glc, they do not survive significantly longer than nonacclimated tips. We have shown that, below a critica1 threshold of glycolytic flux, survival was compromised (Xia et al., 1995). An hypoxic pretreatment induces an increase of maximal in vitro catalytic activities of many enzymes involved in sugar metabolism, a higher ATP level correlated with a higher energy charge (reviewed by Ricard et al., 1994), and a better regulation of cytoplasmic pH linked to an efficient efflux of lactic acid (Xia and Roberts, 1994;Xia et al., 1995;Xia and Saglio, 1992). However, the role of these modifications in the subsequent survival of tissues in anoxia is not obvious. For example, we have shown that in HPT root tips the level of ATP is not critica1 for survival or for cytosolic pH regulation in anoxia (Xia et al., 1995), and the increase in glycolytic enzyme activities is not correlated with a higher glycolytic rate during the 90 min after the transfer of tissues from air to strictly anoxic conditions. During this period, the glycolytic rate (measured as the sum of ethanol plus lactate) remains strictly identical in HPT and NHPT * Corresponding author; e-mail saglio@bordeaux.inra.fr; fax 56-84-32-45. root tips. It is only after 60 to 90 min that the glycolytic flux declines and then almost stops in NHPT root tips even in the presence of added Glc, whereas it remains sustained in HPT tips (Xia and Saglio, 1992). Such a decline of ethanol production during the hours or days after the transfer to anoxia appears to be common to many plants (Smith and ap Rees, 1979;Raymond et al., 1985; Kimmerer and Mac Donald, 1987). Exceptions are rice seeds and seedlings, which are resistant to anoxia and in which the rate of ethanol production increases during at least the first 2 d in the absence of O, (Raymond et al., 1985).How glycolytic flux is modified in HPT and NHPT root tips and what is responsible for its decline in NHPT tips early in anoxia (...

Carbon Partitioning during Sucrose Accumulation in Sugarcane Internodal Tissue

“…However, it is evident from the flux analysis (Table IV) that mobilization of both Glc and Fru decreased in the more mature tissue and it might therefore imply that a more complex regulation than only hexose availability limits the in vivo activity of these enzymes. Hexokinases have previously been implicated in the regulation of glycolytic flux in plants (Renz andStitt, 1993a, 1993b;Bouny and Saglio, 1996). In animal tissues hexokinases also play an important role in controlling glycolytic flux (German, 1993;Kashiwaya et al, 1994) and can have control coefficients as high as 0.7 to 0.9 (Rapoport et al, 1974).…”

Oxygen Sensing via the Ethylene Response Transcription Factor RAP2.12 Affects Plant Metabolism and Performance under Both Normoxia and Hypoxia

“…After addition of 1 mL 100% ethanol, the NADH production was measured photometrically at 340 nm. PDC and LDH activity measurements were performed according to Bouny and Saglio (1996), with extraction of 20 mg frozen plant powder in 500 mL extraction buffer for PDC activity determination, and in 300 mL extraction buffer for LDH activity quantification. In each case, 30 mL amounts of the supernatant were mixed with 170 mL reaction buffer for measurement.…”

The Role of Sugars, Hexokinase, and Sucrose Synthase in the Determination of Hypoxically Induced Tolerance to Anoxia in Tomato Roots

“…The low activity found for PDC in aerobic tissues and its substantial induction in hypoxia suggested that its activity might be limiting in nonacclimated tissues (Waters et al, 1991). However, PDC is also strongly activated by the low cytosolic pH (Davies et al, 1974) usually found in nonacclimated anoxic tissues (Xia et al, 1995;Bouny and Saglio, 1996), and hypoxic induction of PDC may not, therefore, be necessary to allow anoxic tolerance.…”

“…Induction of lactic acid excretion 1 has been correlated with better survival of acclimated tissues (Xia and Saglio, 1992;Rivoal and Hanson, 1993), but the importance of excretion in cytosolic pH regulation is doubtful (Xia and Roberts, 1996), and whether lactic acid accumulation alone accounts for cytosolic acidosis is still a matter of debate (Roberts et al, 1984a;Saint-Gès et al, 1991). Among the proteins induced during hypoxic acclimation, only HKs have been demonstrated to contribute to the survival of acclimated maize root tips (Bouny and Saglio, 1996) by allowing the maintenance of a sustained glycolytic rate. Induction of ADH (Andrews et al, 1994) does not appear to be related to anoxic tolerance.…”