<sup>31</sup>P-NMR Spectroscopy of Roots of Intact Corn Seedlings

Roberts, Justin K. M.; Testa, M.

doi:10.1104/pp.86.4.1127

Cited by 16 publications

(6 citation statements)

References 2 publications

(2 reference statements)

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“…6, A and B). These spectroscopic signatures are characteristic of living root tips (Roberts and Testa, 1988), and confirm the viability measurements in Figure 4. In contrast, roots that had been exposed to 10 m cycloheximide during hypoxic pretreatment lost essentially all of these spectroscopic signatures (Fig.…”

Section: Inhibition Of Protein Synthesis During Hypoxic Acclimation Csupporting

confidence: 72%

Patterns of protein synthesis and tolerance of anoxia in root tips of maize seedlings acclimated to a low-oxygen environment, and identification of proteins by mass spectrometry

2000

Current Opinion in Plant Biology

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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.

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Section: Inhibition Of Protein Synthesis During Hypoxic Acclimation Csupporting

confidence: 72%

Patterns of protein synthesis and tolerance of anoxia in root tips of maize seedlings acclimated to a low-oxygen environment, and identification of proteins by mass spectrometry

2000

Current Opinion in Plant Biology

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“…Maize seeds (Zea mays L., inbred B73, a kind gift from Pioneer Hi-Bred International, Johnston, IA) were germinated and grown until the seedlings had primary roots approximately 10 cm long as described previously (Roberts and Testa, 1988). Groups of 25 seedlings were then placed in a glass funnel, and the stem was inserted in a polypropylene tube (Poly-Prep column, Bio-Rad) so that the primary roots could be immersed in 15 mL of basal medium 10.7 mM KNO,,0.65 mM &(NO,) [v/vl O, in N, for HPT; in both cases without pre-equilibration) through the bottom of the tube.…”

Section: Materials a N D Methodsmentioning

confidence: 99%

“…Seedlings were subjected to pretreatments and anoxia as in the NMR experiments. After various periods of anoxia, roots of individual seedlings were placed in glass tubes each lined with a strip of filter paper wetted with 0.1 mM CaSO, (Roberts and Testa, 1988); these seedlings were then transferred to a humidified tank (exposed to laboratory fluorescent lights at 23 & 1.5"C), and the elongation after 24 h in air was measured.…”

Section: Materials a N D Methodsmentioning

confidence: 99%

Nucleotide Levels Do Not Critically Determine Survival of Maize Root Tips Acclimated to a Low-Oxygen Environment

1995

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We tested the hypothesis that ATP levels and energy charge determine the resistance of maize (Zea mays) root tips to anoxia. We focused on root tips of whole maize seedlings that had been acclimated to low O , by exposure to an atmosphere of 3% (v/v) O, in N,. Acclimated anoxic root tips characteristically have higher ATP levels and energy charge and survive longer under anoxia than nonacclimated tips. We poisoned intact, acclimated root tips with either fluoride or mannose, causing decreases in ATP and energy charge to values similar to or, i n most cases, below those found in nonacclimated anoxic tips. with the exception of the highest fluoride concentration used, the poisoned, acclimated tips remained much more tolerant of anoxia than nonacclimated root tips. We conclude that high ATP and energy charge are not components critical for the survival of acclimated root tips during anoxia. The reduced nucleotide status in poisoned, acclimated root tips had little effect on cytoplasmic pH regulation during anoxia. This result indicates that i n anoxic, acclimated root tips either cytoplasmic pH regulation is not dominated by ATP-dependent processes or these processes can continue i n vivo largely independently of any changes i n ATP levels i n the physiological range. The role of glycolytic flux in survival under anoxia is discussed.Evaluation of the relative importance of biochemical parameters as determinants of plant performance under environmental stresses, such as low O,, is complicated by the fact that stresses disturb whole networks of biochemical processes (reviewed by Kennedy et al., 1992;Perata and Alpi, 1993;Ricard et al., 1994). Many observed differences between stressed and unstressed, or tolerant and intolerant, plants may well be manifestations of this general disturbance rather than revelations of fundamental processes responsible for the behavior of a particular plant in different environments.The primary biochemical effect of low O, on plant cells is an inhibition of respiratory ATP synthesis, leading to a switch to a lower overall rate of ATP synthesis via fermentation (Beevers, 1961). Given the central role of ATP in cell function (Stryer, 19881, the "energy crisis" in plant cells evoked by low O, would appear to be the direct cause of the eventual loss of function. The resistance of maize root tips to anoxia can be enhanced by pretreatment of intact seedlings with hypoxia ( 2 4 % O,) (Saglio et al., 1988;Johnson et al., 1989). Associated with this acclimation response is the ability to maintain a high glycolytic rate during long periods of anoxia (Saglio et al., 1988;Hole et al., 1992;Xia and Saglio, 1992) and higher ATP levels and energy charge (Saglio et al., 1988;Johnson et al., 1994). One straightforward interpretation of this phenomenon is that the primary acclimation response leading to improved tolerance of anoxia is the enhanced energy metabolism, which relieves the shortage of ATP and facilitates cell function. Moreover, reported differences between acclimated and nonacclimated root t...

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“…Maize seeds (Zea Mays L., inbred B73, a kind gift from Pioneer Hi-Bred International, Johnston, IA) were germinated and grown until the seedlings had primary roots approximately 10 cm long as described previously (Roberts and Testa 1988). Groups of 25 seedlings were then placed in a special modified glass funnel similar to that in Figure 1, with their primary roots immersed in a nutrient solution according to Xia et al (1995).…”

Section: Materials a N D M E T H O D Smentioning

confidence: 99%

“…The increase of the medium pH throughout the experiment was less than 0.1 pH unit. After various periods of anoxia, roots of individual seedlings were placed in glass tubes, each lined with a strip of filter paper wetted with 0.1 mM CaSO, (Roberts and Testa, 1988); these seedlings were then transferred to a humidified tank (exposed to laboratory fluorescent lights at 23 ? 1.5"C), and the elongation after 24 h in air was measured.…”

Section: Materials a N D M E T H O D Smentioning

confidence: 99%

Regulation of H+ Extrusion and Cytoplasmic pH in Maize Root Tips Acclimated to a Low-Oxygen Environment

Xia

Roberts

1996

Plant Physiology

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We tested the hypothesis that H+ extrusion contributes to cytoplasmic pH regulation and tolerance of anoxia in maize (Zea mays) root tips. We studied root tips of whole seedlings that were acclimated to a low-oxygen environment by pretreatment in 3% (v/v) O,. Acclimated root tips characteristically regolate cytoplasmic pH near neutrality and survive prolonged anoxia, whereas nonacclimated tips undergo severe cytoplasmic acidosis and die much more quickly. We show that the plasma membrane H+-AlPase can operate under anoxia and that net H+ extrusion increases when cytoplasmic pH falls. However, at an external pH near 6.0, H+ extrusion contributes little to cytoplasmic pH regulation. At more acidic external p H values, net H+ flux into root tips increases dramatically, leading to a decrease in cytoplasmic pH and reduced tolerante of anoxia. We present evidence that, under these conditions, H+ pumps are activated to partly offset acidosis due to H+ influx and, thereby, contribute to cytoplasmic pH regulation and tolerante of anoxia. l h e regulation of H+ extrusion under anoxia is discussed with respect to the acclimation response and mechanisms of intracellular p H regulation in aerobic plant cells.Root tips of whole maize (Zea mays) seedlings subjected to HPT with 3% (v/v) O, in N, survive longer under anoxia than NHPT tips (Saglio et al., 1988;Johnson et al., 1989). HPT root tips regulate cytoplasmic pH near neutrality for many hours, in contrast to NHPT root tips, which exhibit severe cytoplasmic acidosis early in anoxia (Xia and Roberts, 1994;Xia et al., 1995). Cytoplasmic acidosis can cause early death in hypoxic maize root tips (Roberts et al., 1984), and the extent of cytoplasmic acidosis in plants under low oxygen has been widely shown to correlate with their intolerance of this stress (reviewed by Kennedy et al., 1992 andRicard et al., 1994). It is therefore important to understand the factors and mechanisms contributing to intracellular pH regulation in plant cells under anoxia.One phenomenon that may be valuable in preventing cytoplasmic acidosis during anoxic stress is lactate efflux, which has been observed to be induced in maize roots during acclimation in 3% O, (Xia and Saglio, 1992;Xia and Roberts, 1994) and also in certain Limonium sp. (Rivoal and Hanson, 1993). Fermentation to cytoplasmic lactate leads to significant cytoplasmic acidosis (reviewed by Ricard et al., 1994). Therefore, it is possible that remova1 of cytoplasmic lactate by efflux might serve to limit acidosis. Such a pos- sibility depends in part on the fate of H+ generated during fermentation of Glc to lactate; H+ generation is stoichiometric with lactate at constant ATP (Hochachka and Mommsen, 1983). It is not currently known if HPT root tips excrete lactate anion or lactic acid or if cytoplasmic H+ produced during fermentation to lactate is pumped into the extracellular space or the vacuole.Saint-Ges et al. (1991) observed a correlation between cytoplasmic pH and ATP levels after the onset of anoxia in excised NHPT maize root tips....

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³¹P-NMR Spectroscopy of Roots of Intact Corn Seedlings

Cited by 16 publications

References 2 publications

Patterns of protein synthesis and tolerance of anoxia in root tips of maize seedlings acclimated to a low-oxygen environment, and identification of proteins by mass spectrometry

Patterns of protein synthesis and tolerance of anoxia in root tips of maize seedlings acclimated to a low-oxygen environment, and identification of proteins by mass spectrometry

Nucleotide Levels Do Not Critically Determine Survival of Maize Root Tips Acclimated to a Low-Oxygen Environment

Regulation of H+ Extrusion and Cytoplasmic pH in Maize Root Tips Acclimated to a Low-Oxygen Environment

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31P-NMR Spectroscopy of Roots of Intact Corn Seedlings

Cited by 16 publications

References 2 publications

Patterns of protein synthesis and tolerance of anoxia in root tips of maize seedlings acclimated to a low-oxygen environment, and identification of proteins by mass spectrometry

Patterns of protein synthesis and tolerance of anoxia in root tips of maize seedlings acclimated to a low-oxygen environment, and identification of proteins by mass spectrometry

Nucleotide Levels Do Not Critically Determine Survival of Maize Root Tips Acclimated to a Low-Oxygen Environment

Regulation of H+ Extrusion and Cytoplasmic pH in Maize Root Tips Acclimated to a Low-Oxygen Environment

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³¹P-NMR Spectroscopy of Roots of Intact Corn Seedlings