Contribution of Malate and Amino Acid Metabolism to Cytoplasmic pH Regulation in Hypoxic Maize Root Tips Studied Using Nuclear Magnetic Resonance Spectroscopy

³¹P-, ¹³C-, and ¹⁵N-nuclear magnetic resonance spectroscopy were used to determine the roles of malate, succinate, Ala, Asp, Glu, Gln, and γ-aminobutyrate (GABA) in the energy metabolism and regulation of cytoplasmic pH in hypoxic maize (Zea mays L.) root tips. Nitrogen 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 Gln, 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 Glc 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 patterns of metabolism during hypoxia is considered with respect to cytoplasmic pH and redox state.     

Dependence of Ethanolic Fermentation, Cytoplasmic pH Regulation, and Viability on the Activity of Alcohol Dehydrogenase in Hypoxic Maize Root Tips

We examined the role of alcohol dehydrogenase (ADH) in the metabolism and survival of hypoxic maize (Zea mays L.) root tips. The dependence of the rate of ethanolic fermentation, cytoplasmic pH, and viability on the activity of ADH in maize root tips during extreme hypoxia was determined. Maize lines with ADH activities differing over about a 200-fold range were studied. Effects of genetic background were controlled by comparing pairs of F4 progeny of crosses between mutant (low ADH activity) and reference inbred lines. The capacity of hypoxic root tips to perform ethanolic fermentation exhibited a dependence on ADH activity only at activities found in Adh 1 nulls. The ability of maize root tips to withstand prolonged and extreme hypoxia was like-wise independent of ADH activity, except at the lowest activities. Root tips that exhibited lower tolerance of hypoxia had more acidic cytoplasm during extreme hypoxia. We conclude that the activity of ADH in normal maize root tips does not limit the capacity for energy production via fermentation, and does not determine viability under extreme hypoxia. The significance of the induction of ADH activity in plants by hypoxia is discussed.     

Further Evidence that Cytoplasmic Acidosis Is a Determinant of Flooding Intolerance in Plants

We present two pieces of evidence that regulation of cytoplasmic pH near neutrality is a prerequisite for survival of root tips during hypoxia. First, blackeye peas and navy beans show earlier cytoplasmic acidosis under hypoxia than soybeans or pumpkin or maize, and die earlier. Second, when cytoplasmic acidosis in maize root tips is greatly retarded by treatment with 25 millimolar Ca(NO₃)₂, they remain viable under hypoxia for a much longer period of time than untreated hypoxic root tips. We also show that viability of maize root tips is unaffected by the supply of exogenous sugar (and so on the rate of ethanolic fermentation) for at least 16 hours of hypoxia.     

Hypoxia does not affect rate of ATP synthesis and energy metabolism in rice shoot tips as measured by 31P NMR in vivo.

The cytoplasmic pH, concentrations of phosphate metabolites, and rate of ATP synthesis were measured in vivo in excised rice shoot tips under normoxic and hypoxic conditions using 31P NMR. When supplied with glucose, the shoot tips grew rapidly and were relatively unaffected by hypoxia. The cytoplasmic pH decreased transiently by only 0.2 units during hypoxia, and the concentration of ATP was maintained to at least 90% of the normoxic level. Most importantly, the unidirectional rate constant of ATP synthesis from free phosphate decreased less than 25% during hypoxia. This is in contrast to other actively growing tissues such as the maize root tip. gamma-Aminobutyrate was the major nonvolatile fermentation end product after 22 h of hypoxia. Other hypoxia-induced changes included a modest increase in [Ala] and [succinate] as well as a substantial decrease in [malate].     

"GROWTH-PROMOTING SUBSTANCE" AND ELONGATION OF ROOTS

The vertical elongation of normal roots of Lupinus seedlings proceeds at constant rate over periods of 4 to 5 hours. The decapitation of a root stops its elongation for a variable length of time, followed by a period of renewed elongation at a rate lower than that of the normal root. The tipping of the decapitated root with a tip of a coleoptile of Zea induces a decrease in the rate of elongation of the root. The same effect can be obtained with the diffusate from tips of coleoptile of Avena and to a lesser extent with diffusate of root tips. The reduction in the rate of elongation of the root determined by diffusate from the lower half of the tip of a coleoptile placed horizontally is more pronounced than the inhibition elicited by the diffusate of the upper half of the same tip. Various experiments with the diffusate of tips support the idea that under the conditions used the growth-promoting substance of the coleoptile tip or root tip inhibits the elongation of the decapitated root.     

Observation of Cytoplasmic and Vacuolar Malate in Maize Root Tips by C-NMR Spectroscopy

The accumulation of malate by maize (Zea mays L.) root tips perfused with KH¹³CO₃ was followed by ¹³C nuclear magnetic resonance spectroscopy. In vivo nuclear magnetic resonance spectra contained distinct signals from two pools of malate in maize root tips, one at a pH ~5.3 (assigned to the vacuole) and one at a pH > 6.5 (assigned to the cytoplasm). The ratio of cytoplasmic to vacuolar malate was lower in 12 millimeter long root tips than in 2 millimeter root tips. The relatively broad width of the signals from C1- and C4-labeled vacuolar malate indicated heterogeneity in vacuolar pH. During the 3 hour KH¹³CO₃ treatment, ¹³C-malate accumulated first primarily in the cytoplasm, increasing to a fairly constant level of ~6 millimolar by 1 hour. After a lag, vacuolar malate increased throughout the experiment.     

Isolation of polyribosomes and messenger RNA active in in vitro synthesis of soybean seed proteins

Polyribosome preparations containing low proportions of monosomes to polyribosomes have been isolated from developing seeds of Glycine max L. Merrill using a high pH-high KCl buffer. The polyribosomes were functional in in vitro protein synthesis reactions using wheat germ 23,000g supernatant preparations. Results of experiments using aurintricarboxylic acid indicated that most or all of the amino acid incorporation in vitro resulted from the completion of nascent polypeptides associated with the isolated polyribosmes. RNA purified from polyribosome preparations by affinity chromatography on oligo(dT)-cellulose was also active in vitro, and had different Mg and K requirements for translation than did the polyribosomes. Translation of oligo(dT)-cellulose-purified mRNA was inhibited by the addition of 7-methylguanosine 5′-phosphate, suggesting that soybean mRNAs are “capped” at their 5′ ends. Some, but not all, of the products of these reactions were identical in electrophoretic mobility to radioactive polypeptides of storage proteins produced in soybean cotyledons grown in culture.     

Synthesis and deposition of zein in protein bodies of maize endosperm

The origin of protein bodies in maize (Zea mays L.) endosperm was investigated to determine whether they are formed as highly differentiated organelles or as protein deposits within the rough endoplasmic reticulum. Electron microscopy of developing maize endosperm cells showed that membranes surrounding protein bodies were continuous with rough endoplasmic reticulum membranes. Membranes of protein bodies and rough endoplasmic reticulum both contained cytochrome c reductase activity indicating a similarity between these membranes. Furthermore, the proportion of alcohol-soluble protein synthesized by polyribosomes isolated from protein body or rough endoplasmic reticulum membranes was similar, and the alcohol-soluble or -insoluble proteins showed identical [¹⁴C]leucine labeling. These results demonstrated that protein bodies form simply as deposits within the rough endoplasmic reticulum.

Messenger RNA that directed synthesis of only the smaller molecular weight zein subunit was separated from mRNA that synthesized both subunits by sucrose gradient centrifugation. This result demonstrated that separate but similar sized mRNAs synthesize the major zein components. In vitro translation products of purified mRNAs or polyribosomes were approximately 2,000 daltons larger than native zein proteins, suggesting that the proteins are synthesized as zein precursors. When intact rough endoplasmic reticulum was placed in the in vitro protein synthesis system, proteins corresponding in molecular weight to the native zein proteins were obtained.

     

Sites of gibberellin biosynthesis in pea seedlings

Potential sites of gibberellin biosynthesis in 10-day-old `Alaska' pea (Pisum sativum L.) seedlings were investigated using a cell-free ezyme system capable of incorporating [¹⁴C]-mevalonic acid into ent-kaurene. In peas, ent-kaurene is assumed to be a committed intermediate in the gibberellin biosynthetic pathway. Comparative results from enzyme assays using extracts from shoot tips, leaf blades, internodes, and root tips indicate that the highest capacity for ent-kaurene (and presumably gibberellin) synthesis is in those tissues with the greatest potential for growth. The highest rates were obtained with extracts prepared from the fifth (youngest) internode, the fourth (youngest) expanded leaf, and the shoot tip itself. This report represents the first direct evidence that the enzymes responsible for early stages in gibberellin biosynthesis occur in internode tissues with potential for rapid elongation.     

Activation and stabilization of nitrate reductase by NADH in wheat and maize

Preincubation of nitrate reductase (NR) extracted from wheat shoot tips with NADH in vitro, activated and stabilized activity at both O° and 25°. However, preincubation with potassium ferricyanide inactivated the NR in vitro. NADH also stabilized the NR activity in extracts from maize shoot tips. It was observed that NR from both wheat and maize was active at low temperatures.     

Improved Cytoplasmic pH Regulation,Increased Lactate Efflux,and Reduced Cytoplasmic Lactate Levels Are Biochemical Traits Expressed in Root Tips of Whole Maize Seedlings Acclimated to a Low-Oxygen Environment

We tested the hypothesis (J.-H. Xia and P.H. Saglio [1992] Plant Physiol 100: 40-46) that the enhanced ability of maize (Zea mays) root tips to survive anoxia, elicited by a 4-h exposure to 3% O2 ("acclimation"), is due to less cytoplasmic acidosis early in anoxia. Cytoplasmic pH and fermentation reactions were monitored in excised and intact (attached) maize root tips by simultaneous in vivo 13C- and 31P-NMR spectroscopy. We demonstrate that both excised and intact acclimated root tips have significantly higher cytoplasmic pH values under anoxia. This reduction in cytoplasmic acidosis is greater in intact root tips. Remarkably, cytoplasmic pH does not change when root tips are transferred from 3% O2 to anoxia. The earlier observation of considerable lactate efflux and lowered intracellular lactate in excised, acclimated root tips (ibid.) was extended to intact seedlings. The predominant fermentation end product retained in the cells of acclimated root tips is alanine. We discuss the relationship between cytoplasmic pH and levels of intracellular lactate and alanine in sugar-replete roots, and the role of cytoplasmic pH in determining survival under anoxia.     

Contribution of Malic Enzyme, Pyruvate Kinase, Phosphoenolpyruvate Carboxylase, and the Krebs Cycle to Respiration and Biosynthesis and to Intracellular pH Regulation during Hypoxia in Maize Root Tips Observed by Nuclear Magnetic Resonance Imaging and Gas Chromatography-Mass Spectrometry

In vivo pyruvate synthesis by malic enzyme (ME) and pyruvate kinase and in vivo malate synthesis by phosphoenolpyruvate carboxylase and the Krebs cycle were measured by ¹³C incorporation from [1-¹³C]glucose into glucose-6-phosphate, alanine, glutamate, aspartate, and malate. These metabolites were isolated from maize (Zea mays L.) root tips under aerobic and hypoxic conditions. ¹³C-Nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry were used to discern the positional isotopic distribution within each metabolite. This information was applied to a simple precursor-product model that enabled calculation of specific metabolic fluxes. In respiring root tips, ME was found to contribute only approximately 3% of the pyruvate synthesized, whereas pyruvate kinase contributed the balance. The activity of ME increased greater than 6-fold early in hypoxia, and then declined coincident with depletion of cytosolic malate and aspartate. We found that in respiring root tips, anaplerotic phosphoenolpyruvate carboxylase activity was high relative to ME, and therefore did not limit synthesis of pyruvate by ME. The significance of in vivo pyruvate synthesis by ME is discussed with respect to malate and pyruvate utilization by isolated mitochondria and intracellular pH regulation under hypoxia.     

Eukaryotic Polyribosome Profile Analysis

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase or the elongation phase by a variety of cellular stresses such as amino acid starvation or growth factor withdrawal. Alternatively, translation of individual mRNAs can be regulated by mRNA localization or the presence of cognate microRNAs. Studies of protein synthesis frequently utilize polyribosome analysis to shed light on the mechanisms of translation regulation or defects in protein synthesis. In this assay, mRNA/ribosome complexes are isolated from eukaryotic cells. A sucrose density gradient separates mRNAs bound to multiple ribosomes known as polyribosomes from mRNAs bound to a single ribosome or monosome. Fractionation of the gradients allows isolation and quantification of the different ribosomal populations and their associated mRNAs or proteins. Differences in the ratio of polyribosomes to monosomes under defined conditions can be indicative of defects in either translation initiation or elongation/termination. Examination of the mRNAs present in the polyribosome fractions can reveal whether the cohort of individual mRNAs being translated changes with experimental conditions. In addition, ribosome assembly can be monitored by analysis of the small and large ribosomal subunit peaks which are also separated by the gradient. In this video, we present a method for the preparation of crude ribosomal extracts from yeast cells, separation of the extract by sucrose gradient and interpretation of the results. This procedure is readily adaptable to mammalian cells.     

The Correlation of Profiles of Surface pH and Elongation Growth in Maize Roots

High-resolution profiles of surface pH and growth along vertically growing maize (Zea mays) primary root tips were determined simultaneously by pH-sensitive microelectrodes and marking experiments. Methodological tests were carried out that proved the reliability of our kinematic growth analysis, while questioning the validity of an alternative technique employed previously. A distal acidic zone around the meristematic region and a proximal one around the elongation zone proper were detected. This pattern as such persisted irrespective of the bulk pH value. The proximal acidic region coincided with maximum relative elemental growth rates (REGR), and both characters reacted in a correlated manner to auxin and cyanide. The distal acidic band was unrelated to growth, but was abolished by cyanide treatment. We conclude that: (a) the pattern of surface pH as such is a regulated feature of growing root tips; (b) the correlation of extracellular pH and growth rate suggests a functional relationship only along proximal portions of the growing root tip; and (c) the distal acidic band is not caused by pH buffering by root cap mucilage, as suggested previously, but rather is controlled by cellular activity.     

Kinetic studies of the variations of cytoplasmic pH, nucleotide triphosphates (31P-NMR) and lactate during normoxic and anoxic transitions in maize root tips

We have followed the dynamic evolution of intracellular pH and of the intracellular concentration of nucleotides (NDP, NTP), Pi and lactate in maize root tips during the course of normoxia and anoxia transition. The intracellular pH, determined from the 31P-NMR chemical shift of the cytoplasmic P1 peak, dropped from 7.5 to 6.9 during the first few minutes after anaerobiosis. It increased again, then settled to a steady-state value of 7.1-7.2, 25 min after the beginning of the anoxic treatment. Following oxygenation, the chemical shift of the cytoplasmic Pi peak drifted gradually to its initial value. The cytoplasmic pH followed an oscillatory time course which was almost identical to the time course of NTP. Intracellular lactate accumulated steadily during the first 30 min after anaerobiosis, then its intracellular concentration remained almost constant. Following oxygenation, the intracellular concentration of lactate decreased slowly. The cytoplasmic pH followed a time course which was not identical to the time course of lactate. Following hypoxia, the pH dropped to low values long before the intracellular lactate concentration reached a steady-state equilibrium. Conversely, subsequent to oxygenation, the pH returned to normal values long before lactate. These results do not agree with the statement that cytoplasmic acidification in hypoxic maize root tips is necessarily associated with lactic acid synthesis.     

Anoxic Responses of Seedling Roots of Rice Cultivars Varying in Tolerance to Submergence

Differences in tolerance to submergence and anoxia exhibited by cultivar-specific rice (Oryza sativa L.) extend to the primary root tips and axes of 3-day-old seedlings. This paper considers the physiological mechanisms which might account for rice root intolerance to anoxia, particularly those implicated in pH regulation and sugar metabolism in relation to hypoxic acclimation. Hypoxic treatment and the presence of glucose during anoxia did not permit root tips and axes of intolerant cultivars to survive 24-h anoxia. The absence of typical glycolytic and fermentative enzyme induction together with no improvement of ethanol production and energy status during anoxia suggest that intolerant cultivars are not capable of hypoxic acclimation at the level of energy and sugar metabolism. However, root tip survival was enhanced in buffered medium after hypoxic treatment, suggesting a relationship between hypoxic treatment and improved pH regulation.     

Hardening of root cell walls: a growth inhibitory response to salinity stress

Primary roots of intact maize plants (Zea mays L.) grown for several days in nutrient solutions containing 100 mol m⁻³ NaCl and additional calcium, had relatively inhibited rates of elongation. Possible physical restraints underlying this salt induced inhibition were investigated. The inhibition did not involve reductions in osmotic potential gradients and turgor in the tip tissues responsible for root elongation growth. The apparent yield threshold pressure, which is related to capacity of cell walls to undergo loosening by stress relaxation, was estimated psychrometrically in excised root tips. Salinity increased yield threshold values. Comparative root extensibility values were obtained for intact plants by determining the initial (1 min) increase in root elongation rate induced by an 0.1 MPa osmotic jump. Comparative extensibility was significantly reduced in the salinized root tips. Salinity did not reduce capacities for water efflux and associated elastic contraction in root tip tissues of intact plants exposed to hypertonic mannitol. We conclude that cell wall hardening in the elongating root tips is an important component of root growth inhibition induced by long-term salinization.     

Regulation of Cytoplasmic and Vacuolar pH in Maize Root Tips under Different Experimental Conditions

³¹P-Nuclear magnetic resonance spectra of perfused maize (Zea mays L., hybrid WW x Br 38) root tips, obtained at 10-minute intervals over 12 hours or longer, indicate that no cytoplasmic or vacuolar pH changes occur in these cells in the presence of 25 millimolar K₂SO₄, which induces extrusion of 4 to 5 microequivalents H⁺ per gram per hour. In contrast, hypoxia causes cytoplasmic acidification (0.3-0.6 pH unit) without a detectable change in vacuolar pH. The cytoplasm quickly returns to its original pH on reoxygenation. Dilute NH₄OH increases the vacuolar pH more than it does the cytoplasmic pH; after NH₄OH is removed, the vacuole recovers its original pH more slowly than does the cytoplasm. The results indicate that regulation of cytoplasmic pH and that of vacuolar pH in plant cells are separate processes.     

Asparagine synthetase in corn roots

The level of asparagine synthetase is low in 10-mm root tips from corn seedings (Zea mays W64 × W182F) but relatively high in mature root sections taken 20 to 35 mm from the tip. When root tips are excised there is a marked increase in asparagine synthetase over a 5-hour period. In mature root sections, on the other hand, the asparagine synthetase activity declines over the same 5-hour period. The increase in the root tip is sensitive to cordycepin, 6-methylpurine, and cycloheximide, which indicates that both RNA and protein synthesis are involved in the formation of asparagine synthetase in the root tip sections. The glutamine analogue azaserine also inhibits formation of the enzyme in root tips, as does glucose. The increase in the root tip is not sensitive to asparagine. Additions of glucose or asparagine have no effect on enzyme activity in extracts. When cycloheximide, azaserine, or glucose is added to the mature root sections there is no effect on recovered enzyme activity.     

Intracellular pH in rice and wheat root tips under hypoxic and anoxic conditions

The influence of anoxia and hypoxia on dynamic of intracellurar pH and ATP content in rice and wheat root tips was investigated with ³¹P-NMR spectroscopy. Both cereals responded to hypoxia similarly, by rapid cytoplasmic acidification (from pH 7.6–7.7 to 7.1), which was followed by slow partial recovery (0.3 units). Anoxia led to a dramatic pH_cyt drop in tissues of both species (from pH 7.6–7.7 to less than 7.0) and partial recovery took place in rice only. In wheat, the acidification continued to pH 6.8 after 6 h of exposure. Anoxic wheat root tips were deficient in ADH induction, whereas increased activity of alcoholic fermentation enzymes took place in anoxic rice root tips, as well as in both species after hypoxic treatment. In both plants, NTP content followed the dynamics of pH_cyt. There was a strong correlation between NTP content and cytoplasmic H⁺ activity ([H⁺]_cyt = 10^−pHcyt) for both hypoxic and anoxic conditions. In this addendum we want to focus the reader''s attention on the importance of adequate experimental design when hypoxia is under investigation and on some further perspectives of intracellular pH regulation in plants under anaerobic conditions.Key words: anoxia, hypoxia, rice, wheat, cytoplasmic pH regulation