We show that a transient lactic fermentation provides the signal triggering ethanol production in hypoxic maize root tips. The signal is cytoplasmic pH. This interaction between lactic and ethanolic fermentation permits tight cytoplasmic pH regulation during hypoxia-cytoplasmic pH remaining near neutrality for several hours. Mutant roots unable to synthesize ethanol can neither regulate cytoplasmic pH nor maintain ATP levels during extended periods of hypoxia and, like vertebrate tissues, are less tolerant of hypoxia than normal maize. This indicates that cytoplasmic pH regulation is an important factor in survival under hypoxia.Certain higher plant tissues, such as maize roots, although requiring oxygen for normal functioning, can survive long periods (>18 hr) of anaerobiosis (1), with glycolysis continuing during most of this period (2,3). Most vertebrate tissues, on the other hand, can survive only short periods of hypoxia (<2 hr), after which glycolysis is greatly inhibited, and in most cases irreversible cell damage occurs (4,5). This difference between plants and animals cannot be due simply to the lower metabolic rates often seen in plants: the maize root tips (at 250C) used in this study respire five times the rate of a resting man (at 370C) and about half the rate of a resting mouse (at 370C) (6). It is possible, however, that the ability of higher plants to undergo a mainly ethanolic fermentation (2, 3, 7-9), rather than the exclusively lactic fermentation seen in higher animals, is to some degree responsible for their ability to withstand long periods of hypoxia. We considered this view after showing that the cytoplasmic pH of maize root tips falls to a stable value -0.5 pH unit below aerobic values within ""20 min after transfer to an anaerobic environment (10). This behavior is in complete contrast to active, hypoxic vertebrate tissues, where cytoplasmic pH falls throughout hypoxia because of continuous lactic acid accumulation (4, 11, 12) until glycolysis ceases. Thus, in hypoxic vertebrate tissues, energy production (glycolysis) leads to cytoplasmic acidosis, which eventually inhibits continued energy production; in plants, continued energy production does not involve generation of acid (other than carbon dioxide), and so no inhibition of glycolysis due to acidosis is observed.We describe here the mechanism by which cytoplasmic pH is regulated, and ethanol production induced, in hypoxic maize root tips. The mechanism is consistent with in vitro data (13,14). We also show that root tips of mutants that lack the ability to make ethanol during hypoxia-and have decreased viability-cannot regulate cytoplasmic pH but instead, like vertebrate tissues, undergo cytoplasmic acidification throughout hypoxia. MATERIALS AND METHODSExperiments were performed with -=1.5-g samples of 2-mm hybrid maize (WW x Br38) (obtained from Customaize Research, Decateur, IL) root tips excised from 2-day-old seedlings, perfused as described in the figure legends (10). NADH fluorescence (15) was measured in a Perkin-Elm...

Tolerance of anoxia in maize root tips is greatly improved when seedlings are pretreated with 2 to 4 h of hypoxia. We describe the patterns of protein synthesis during hypoxic acclimation and anoxia. We quantified the incorporation of [35 S]methionine into total protein and 262 individual proteins under different oxygen tensions. Proteins synthesized most rapidly under normoxic conditions continued to account for most of the proteins synthesized during hypoxic acclimation, while the production of a very few proteins was selectively enhanced. When acclimated root tips were placed under anoxia, protein synthesis was depressed and no "new" proteins were detected. We present evidence that protein synthesis during acclimation, but not during subsequent anoxia, is crucial for acclimation. The complex and quantitative changes in protein synthesis during acclimation necessitate identification of large numbers of individual proteins. We show that mass spectrometry can be effectively used to identify plant proteins arrayed by two-dimensional gel electrophoresis. Of the 48 protein spots analyzed, 46 were identified by matching to the protein database. We describe the expression of proteins involved in a wide range of cellular functions, including previously reported anaerobic proteins, and discuss their possible roles in adaptation of plants to low-oxygen stress.

We present evidence that cytoplasmic acidosis is a cause of meristematic death in hypoxic root tips of maize and pea seedlings. Usually, leakage of acid from the vacuole is responsible for cytoplasmic acidosis. Leakage of acid, which occurs earlier during hypoxia in pea root tips than in maize root tips, appears to account for the lower tolerance of peas for hypoxia. Cytoplasmic acidosis is accelerated in maize root tips that are either (i) deficient in alcohol dehydrogenase, so that lactic acid production continues throughout hypoxia, or (ii) exposed to external CO2 during hypoxia, or (iii) perfused slowly so that escape of CO2 produced during ethanolic fermentation is retarded. All three conditions decrease the length of time maize root tips can tolerate hypoxia; more rapid cytoplasmic acidosis is associated with more rapid death under hypoxia. Possible mechanisms by which cytoplasmic acidosis leads to death are suggested; the mechanism does not involve inhibition of glycolysis by low pH. It is clear that even species described as "non-flood-tolerant" (9), such as peas, can survive extended periods (days) of flooding (10). Thus, there are no truly flood-intolerant plant species; all plants survive far longer under hypoxia than does any vertebrate (11). We recently presented evidence (11) that the ability of plants to carry out a mainly ethanolic fermentation, rather than the exclusively lactic fermentation seen in higher animals, was responsible for their ability to withstand long periods of hypoxia. In contrast to lactic fermentation, ethanolic fermentation does not result in severe cytoplasmic acidosis (12). Cytoplasmic acidosis appears to be responsible for loss of viability in excised animal organs (13,14). However, our previous experiments (11) did not explain why plant species, all of which apparently carry out a mainly ethanolic fermentation (5, 15-17), differ in their ability to withstand hypoxia. In the study described here we quantitated the ability of excised maize and pea root tips to tolerate extreme hypoxia and measured simultaneously the intracellular pH and the rate of energy production (i.e., rate of ethanolic fermentation) during hypoxia. We present evidence that cytoplasmic acidosis, due to leakage of acid from the vacuole, is a cause of meristematic death in hypoxic root tips. MATERIALS AND METHODSPlant Material. Experiments were carried out using 1-to 2-mm-long root tips from 2-day-old maize (hybrid Funk 4323, obtained from Germain's Seeds, Los Angeles, CA; or F3 progeny, described below) or pea (var. Alaska, from Burpee, Riverside, CA) seedlings. The F3 progeny used in the experiments shown in Fig. 1 were derived from a cross between the maize hybrid, Funk 4343, and plants homozygous for a mutation at the Adhl locus ] that make no active alcohol dehydrogenase-1 (ADH1). Both the normal and the mutant roots contain a low level of ADH2 enzymatic activity, which we previously showed to be insufficient to sustain normal rates of ethanol production and thus permit cytoplasmic pH regulat...

When soybean Glycine max var Wayne seedlings are shifted from a normal growth temperature of 28°C up to 40°C (heat shock or HS), there is a dramatic change in protein synthesis. A new set of proteins known as heat shock proteins (HSPs) is produced and normal protein synthesis is greatly reduced. A brief 10-minute exposure to 45°C followed by incubation at 28°C also results in the synthesis of HSPs. Prolonged incubation (e.g. 1-2 hours) at 45°C results in greatly impaired protein synthesis and seedling death. However, a pretreatment at 40°C or a brief (10-minute) pulse treatment at 45°C followed by a 28C incubation provide protection (thermal tolerance) to a subsequent exposure at 45C. Maximum thermoprotection is achieved by a 2-hour 40°C pretreatment or after 2 hours at 28C with a prior 10-minute 45°C exposure. Arsenite treatment (50 micromolar for 3 hours) also induces the synthesis of HSPlike proteins, and also provides thermoprotection to a 45C HS; thus, there is a strong positive correlation between the accumulation of HSPs and the acquisition of thermal tolerance under a range of conditions. During 40°C HS, some HSPs become localized and stably associated with purified organelle fractions (cg. nuclei, mitochondria4 and ribosomes) while others do not. A chase at 28C results in the gradual loss over a 4-hour period of the HSPs from the organelle fractions, but the HSPs remain selectively localized during a 40C chase period. If the seedlings are subjected to a second HS after a 28C chase, the HSPs rapidly (complete within 15 minute) relocalize in the organelle fractions.The relative amount of the HSPs which relocalize during a second HS increases with higher temperatures from 40°C to 45C. Proteins induced by arsenite treatment are not selectively localized with organelle fractions at 28C but become orpnelle-associated during a subsequent HS at 40°C.The induction of HSPs3 has been shown to be a universal response to thermal stress in a wide range of organisms (5-7, 9, 15, 21, 24, 30, 34 In this report, we present three lines of evidence supporting the role of HSPs in the acquisition of thermotolerance in plants. The criteria for thermoprotection are based on both the growth of soybean seedlings after a 2-h treatment at the lethal temperature of 45°C and the level of amino acid incorporation at this temperature. First, evidence is presented that the two conditions which stimulate the production of HSPs, i.e. a brief exposure to 45°C followed by incubation at 28°C or a somewhat longer exposure to 40°C, provide thermoprotection. Second, some HSPs become selectively localized in cellular organelles during HS and relocalize during a second heat shock after delocalization by a chase at 28°C. A third line of evidence is based on the induction by arsenite of HSP-like proteins. In soybean (results ofthis study) and other systems (5, 14), this respiratory inhibitor stimulates the production of electrophoretically similar proteins that provide thermoprotection and which become localized only during HS in the soyb...

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ABSTRACT31P., 13C-, and "IN-nuclear magnetic resonance spectroscopy were used to determine the roles of malate, succinate, Ala, Asp, Glu, Gin, and y-aminobutyrate (GABA) in the energy metabolism and regulation of cytoplasmic pH in hypoxic maize (Zea mays L.) root tips. Nitogen status was manipulated by perfusing root tips with ammonium sulfate prior to hypoxia; this pretreatment led to enhanced synthesis of Ala early in hypoxia, and of GABA at later times. We show that: (a) the ability to regulate cytoplasmic pH during hypoxia is not significantly affected by enhanced Ala synthesis. (b) Independent of nitrogen status, decarboxylation of Glu to GABA is greatest after several hours of hypoxia, as metabolism collapses. (c) Early in hypoxia, cytoplasmic malate is in part decarboxylated to pyruvate (leading to Ala, lactate, and ethanol), and in part converted to succinate. It appears that activation of malic enzyme serves to limit cytoplasmic acidosis early in hypoxia. (d) Ala synthesis in hypoxic root tips under these conditions is due to transfer of nitrogen ultimately derived from Asp and Gin, present in oxygenated tissue. We describe the relative contributions of glycolysis and malate decarboxylation in providing Ala carbons. (e) Succinate accumulation during hypoxia can be attributed to metabolism of Asp and malate; this flux to succinate is energetically negligible. There is no detectable net flux from GIc to succinate during hypoxia. The significance of the above metabolic reactions relative to ethanol and lactate production, and to flooding tolerance, is discussed. The regulation of the pattems of metabolism during hypoxia is considered with respect to cytoplasmic pH and redox state.

Although synthesis of the cytosolic maize albumin b-32 had been shown to be controlled by the Opaque-2 regulatory locus, its function was unknown. We show here that b-32 is a member of the large and widely distributed class of toxic plant proteins with ribosome-inactivating activity. These ribosome-inactivating proteins (RIPs) are RNA N-glycosidases that remove a single base from a conserved 28s rRNA loop required for elongation factor l u binding. Cell-free in vitro translation extracts were used to show that both maize and wheat ribosomes were resistant to molar excesses of b-32 but not to the dicotyledonous RIP gelonin. We extracted RIP activity from kernels during seed maturation and germination. The amount of RIP activity increased during germination, although the amount of b-32 protein remained fairly constant. Expression of a maize RIP gene under the control of an endosperm-specific transcriptional regulator may be an important clue prompting investigation of the biological basis for RIP expression in seeds of other plants. INTRODUCTIONRibosome-inactivating proteins (RIPs) are a widely distributed group of toxic plant proteins that catalytically inactivate eukaryotic ribosomes (for review, see Stirpe and Barbieri, 1986). RlPs function as N-glycosidases to remove a specific adenine in a conserved loop of the large rRNA . This irreversible modification renders the ribosome unable to bind elongation factor l a , thereby blocking translation. Because this translational inhibitory activity is toxic, RlPs have been tested extensively for use as immunotoxins and antiviral agents and more recently for their effects on protozoa, insects, and fungi (Barbieri and Stirpe, 1982;Cenini et al., 1988;Gatehouse et al., 1990;Leah et al., 1991).RIP activities have been found in the seed, root, leaf, or sap of more than 50 different plant species (Gasperi-Campani et al., 1985). Two forms of RlPs have been described (Stirpe and Barbieri, 1986). Type 1 RlPs such as pokeweed antiviral protein, trichosanthin, the barley translation inhibitor, and gelonin are monomeric enzymes, each with an approximate M, of 30,000 (Irvin, 1975;Stirpe et al., 1980;Asano et al., 1984;Maraganore et al., 1987;Yeung et al., 1988). Type 2 RlPs such as ricin, abrin, and modeccin are highly toxic heterodimeric proteins, each with an approximate M, of 60,000 in which one polypeptide with RIP activity (A-chain) is linked by a disulfide bridge to a galactose-binding lectin (B-chain; Pihl, 1973, 1982;Stirpe et al., 1978).Type 1 RlPs and the A-chain of type 2 RlPs have basic isoelectric points, and many have signal peptides that are not To whom correspondence should be addressed. present in the mature protein (Stirpe and Barbieri, 1986). Although RlPs share biological activity, they typically exhibit similarities of <50%, and antibodies raised against RlPs seldom cross-react with RlPs from distantly related species (Ready et al., 1988). The maize b-32 protein has homology with severa1 previously characterized RIPs, yet it is a singlechain acidic protein tha...