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Justin K M Roberts - One of the best experts on this subject based on the ideXlab platform.
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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
Plant Physiology, 2000Co-Authors: William W P Chang, Cecelia Webster, Lan Huang, Min Shen, Alma L Burlingame, Justin K M RobertsAbstract: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 [35S]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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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 g
Plant Physiology, 1998Co-Authors: Shaune Edwards, Bichty Nguyen, Justin K M RobertsAbstract: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.
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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
Plant Physiology, 1994Co-Authors: Jianhua Xia, Justin K M RobertsAbstract: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.
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contribution of malate and amino acid metabolism to cytoplasmic ph regulation in hypoxic maize Root Tips studied using nuclear magnetic resonance spectroscopy
Plant Physiology, 1992Co-Authors: Justin K M Roberts, Shaune Edwards, Mark A Hooks, Aaron P Miaullis, Cecelia WebsterAbstract:(31)P-, (13)C-, and (15)N-nuclear magnetic resonance spectroscopy were used to determine the roles of malate, succinate, Ala, Asp, Glu, Gln, and gamma-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.
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hypoxia enhances phosphorylation of eukaryotic initiation factor 4a in maize Root Tips
Journal of Biological Chemistry, 1991Co-Authors: Cecelia Webster, Rebecca L Gaut, Joanne M Ravel, Justin K M RobertsAbstract:We have identified two isoforms of initiation factor 4A (eIF-4A) in maize Root Tips, with distinct isoelectric points and similar molecular mass (approximately 50 kDa). Both isoforms of maize eIF-4A cross-react with antibodies raised against wheat germ eIF-4A, and one of the maize proteins (higher pI isoform) comigrates with purified wheat germ eIF-4A on two-dimensional gels. The two maize eIF-4As were indistinguishable by comparative peptide fingerprint analysis, which also showed a very strong similarity between eIF-4A in maize Roots and wheat germ. Maize eIF-4As copurify with eIF-4F and eIF-(iso)4F on a 7-methyl-GTP-Sepharose affinity column, indicating that they are part of the 5'-cap-binding complex. Two-dimensional gel electrophoresis and immunoblotting of proteins from 32P-labeled maize Root Tips revealed that the lower pI isoform of eIF-4A is phosphorylated. Two-dimensional phosphopeptide maps of trypsin-digested eIF-4A contained one principal phosphorylated fragment; phosphoamino acid analysis indicated phosphorylation of threonine. In oxygenated maize Root Tips, the ratio of phosphorylated to nonphosphorylated eIF-4A is approximately 0.2. This ratio increases to approximately 1 within 20 min following the onset of hypoxia, due to interconversion between the two maize eIF-4A isoforms. The hypoxia-induced phosphorylation of eIF-4A is discussed with respect to metabolic responses, and the translational control of gene expression, in hypoxic plant tissues.
Walter J Horst - One of the best experts on this subject based on the ideXlab platform.
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spatial temporal analysis of polyethylene glycol reduced aluminium accumulation and xyloglucan endotransglucosylase action in Root Tips of common bean phaseolus vulgaris
Annals of Botany, 2016Co-Authors: Maolin Zhang, Walter J Horst, Zhongbao YangAbstract:Background and aims Aluminium (Al) toxicity and drought are two major limiting factors for common bean (Phaseolus vulgaris) production on tropical acid soils. Polyethylene glycol (PEG 6000)-induced osmotic stress (OS) simulating drought stress reduces Al accumulation in the entire Root Tips of common bean by alteration of cell-wall (CW) porosity, which might be regulated by two genes encoding xyloglucan endotransglucosylase/hydrolase, PvXTH9 and PvXTHb The aim of this research was to understand the spatial and temporal regulation of both XTH genes in PEG-mediated Al accumulation in the Root Tips. Methods In this study the spatial and temporal expression patterns of Al-inhibited Root elongation, Al accumulation, XTH gene expression and xyloglucan endotransglucosylase (XET) enzyme action in the Root Tips were analysed under PEG-induced OS by a combination of physiological and molecular approaches such as quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and in situ fluorescence detection of XET in Root Tips. Key results The results showed that Al accumulation, expression of XTH genes and XET action were distinctly reduced in the apical 0-2, 2-7 and 7-12 mm zones under OS, implying a potential regulatory role of XTH genes and XET enzyme in CW Al accumulation in these zones. Conclusions The results provide novel insights into the physiological and molecular mechanisms of CW structure modification as a response of plant Roots to OS, which will contribute to mitigate Al and drought stresses, severely limiting crop yields on acid soils.
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effect of aluminium on lipid peroxidation superoxide dismutase catalase and peroxidase activities in Root Tips of soybean glycine max
Physiologia Plantarum, 1991Co-Authors: Ismail Cakmak, Walter J HorstAbstract:Inhibition of Root elongation and modification of membrane properties are sensitive responses of plants to aluminium. The present paper reports on the effect of AI on lipid peroxidation and activities of enzymes related to production of activated oxygen species. Soybean seedlings (Glycine max L. cv. Sito) were precultured in solution culture for 3–5 days and then treated for 1–72 h with Al (AICI3) concentrations ranging from 10 to 75 μM at a constant pH of 4.1. In response to Al supply, lipid peroxidation in the Root Tips (< 2 cm) was enhanced only after longer durations of treatment. Aluminium-dependent increase in lipid peroxidation was intensified by Fe2+ (FeSO4). A close relationship existed between lipid peroxidation and inhibition of Root-elongation rate induced by Al and/or Fe toxicity and/or Ca deficiency. Besides enhancement of lipid peroxidation in the crude extracts of Root Tips due to Al, the activities of superoxide dismutase (EC 1.15.1.1) and peroxidase (EC 1.11.1.7) increased, whereas catalase (EC 1.11.1.6) activity decreased. This indicates a greater generation of oxygen free radicals and related tissue damage. The results suggest that lipid peroxidation is part of the overall expression of Al toxicity in Roots and that enhanced lipid peroxidation by oxygen free radicals is a consequence of primary effects of Al on membrane structure.
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effect of aluminium on lipid peroxidation superoxide dismutase catalase and peroxidase activities in Root Tips of soybean glycine max
Physiologia Plantarum, 1991Co-Authors: Ismail Cakmak, Walter J HorstAbstract:Inhibition of Root elongation and modification of membrane properties are sensitive responses of plants to aluminium. The present paper reports on the effect of AI on lipid peroxidation and activities of enzymes related to production of activated oxygen species. Soybean seedlings (Glycine max L. cv. Sito) were precultured in solution culture for 3–5 days and then treated for 1–72 h with Al (AICI3) concentrations ranging from 10 to 75 μM at a constant pH of 4.1. In response to Al supply, lipid peroxidation in the Root Tips (< 2 cm) was enhanced only after longer durations of treatment. Aluminium-dependent increase in lipid peroxidation was intensified by Fe2+ (FeSO4). A close relationship existed between lipid peroxidation and inhibition of Root-elongation rate induced by Al and/or Fe toxicity and/or Ca deficiency. Besides enhancement of lipid peroxidation in the crude extracts of Root Tips due to Al, the activities of superoxide dismutase (EC 1.15.1.1) and peroxidase (EC 1.11.1.7) increased, whereas catalase (EC 1.11.1.6) activity decreased. This indicates a greater generation of oxygen free radicals and related tissue damage. The results suggest that lipid peroxidation is part of the overall expression of Al toxicity in Roots and that enhanced lipid peroxidation by oxygen free radicals is a consequence of primary effects of Al on membrane structure.
Mindy L Ehrenfried - One of the best experts on this subject based on the ideXlab platform.
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gene expression profiles for cell wall modifying proteins associated with soybean cyst nematode infection petiole abscission Root Tips flowers apical buds and leaves
Journal of Experimental Botany, 2007Co-Authors: Mark L Tucker, Aimee Burke, Charles Murphy, Vanessa K Thai, Mindy L EhrenfriedAbstract:Changes in transcript accumulation for cell wallmodifying proteins were examined in excised soybean Root pieces colonized by soybean cyst nematodes (SCN), Heterodera glycines, using RT-PCR and soybean Affymetrix GeneChips. Sequence-specific PCR primer pairs were prepared from sequence data for core sequences in the GenBank soybean database and consensus sequences derived from the assembly of soybean ESTs. In addition, to identify previously uncharacterized soybean transcripts, degenerate primers were prepared for conserved motifs in cellulases (endo-1,4-b-glucanases, EGases) and polygalacturonases (PGs) and these were used to amplify segments of transcripts that were then extended with 3# and 5# RACE. Several novel EGase and PG transcripts were identified. Gene expression patterns were determined by real-time RT-PCR for 11 EGases, three expansins (EXPs), 14 PGs, two pectate lyases (PLs), and two xyloglucan endotransglucosylase/hydrolases (XTHs) in soybean Roots inoculated with SCN, non-inoculated Roots, serial dissections of Root Tips, leaf abscission zones, flowers, apical buds, and expanding leaves. A large number of genes associated with cell wall modifications are strongly up-regulated in Root pieces colonized by SCN. However, in contrast to most of the transcripts for cell wall proteins, two XTH transcripts were specifically down-regulated in the colonized Root pieces. Gene expression in serial dissections of Root Tips (0‐2 mm, and 2‐7 mm) and whole Roots indicate that the SCN up-regulated genes are associated with a wide range of developmental processes in Roots. Also of interest, many of the cDNAs examined were
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gene expression profiles for cell wall modifying proteins associated with soybean cyst nematode infection petiole abscission Root Tips flowers apical buds and leaves
Journal of Experimental Botany, 2007Co-Authors: Mark L Tucker, Aimee Burke, Charles Murphy, Vanessa K Thai, Mindy L EhrenfriedAbstract:Changes in transcript accumulation for cell wall-modifying proteins were examined in excised soybean Root pieces colonized by soybean cyst nematodes (SCN), Heterodera glycines, using RT-PCR and soybean Affymetrix GeneChips. Sequence-specific PCR primer pairs were prepared from sequence data for core sequences in the GenBank soybean database and consensus sequences derived from the assembly of soybean ESTs. In addition, to identify previously uncharacterized soybean transcripts, degenerate primers were prepared for conserved motifs in cellulases (endo-1,4-beta-glucanases, EGases) and polygalacturonases (PGs) and these were used to amplify segments of transcripts that were then extended with 3' and 5' RACE. Several novel EGase and PG transcripts were identified. Gene expression patterns were determined by real-time RT-PCR for 11 EGases, three expansins (EXPs), 14 PGs, two pectate lyases (PLs), and two xyloglucan endotransglucosylase/hydrolases (XTHs) in soybean Roots inoculated with SCN, non-inoculated Roots, serial dissections of Root Tips, leaf abscission zones, flowers, apical buds, and expanding leaves. A large number of genes associated with cell wall modifications are strongly up-regulated in Root pieces colonized by SCN. However, in contrast to most of the transcripts for cell wall proteins, two XTH transcripts were specifically down-regulated in the colonized Root pieces. Gene expression in serial dissections of Root Tips (0-2 mm, and 2-7 mm) and whole Roots indicate that the SCN up-regulated genes are associated with a wide range of developmental processes in Roots. Also of interest, many of the cDNAs examined were up-regulated in petiole abscission zones induced to abscise with ethylene.
Ismail Cakmak - One of the best experts on this subject based on the ideXlab platform.
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effect of aluminium on lipid peroxidation superoxide dismutase catalase and peroxidase activities in Root Tips of soybean glycine max
Physiologia Plantarum, 1991Co-Authors: Ismail Cakmak, Walter J HorstAbstract:Inhibition of Root elongation and modification of membrane properties are sensitive responses of plants to aluminium. The present paper reports on the effect of AI on lipid peroxidation and activities of enzymes related to production of activated oxygen species. Soybean seedlings (Glycine max L. cv. Sito) were precultured in solution culture for 3–5 days and then treated for 1–72 h with Al (AICI3) concentrations ranging from 10 to 75 μM at a constant pH of 4.1. In response to Al supply, lipid peroxidation in the Root Tips (< 2 cm) was enhanced only after longer durations of treatment. Aluminium-dependent increase in lipid peroxidation was intensified by Fe2+ (FeSO4). A close relationship existed between lipid peroxidation and inhibition of Root-elongation rate induced by Al and/or Fe toxicity and/or Ca deficiency. Besides enhancement of lipid peroxidation in the crude extracts of Root Tips due to Al, the activities of superoxide dismutase (EC 1.15.1.1) and peroxidase (EC 1.11.1.7) increased, whereas catalase (EC 1.11.1.6) activity decreased. This indicates a greater generation of oxygen free radicals and related tissue damage. The results suggest that lipid peroxidation is part of the overall expression of Al toxicity in Roots and that enhanced lipid peroxidation by oxygen free radicals is a consequence of primary effects of Al on membrane structure.
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effect of aluminium on lipid peroxidation superoxide dismutase catalase and peroxidase activities in Root Tips of soybean glycine max
Physiologia Plantarum, 1991Co-Authors: Ismail Cakmak, Walter J HorstAbstract:Inhibition of Root elongation and modification of membrane properties are sensitive responses of plants to aluminium. The present paper reports on the effect of AI on lipid peroxidation and activities of enzymes related to production of activated oxygen species. Soybean seedlings (Glycine max L. cv. Sito) were precultured in solution culture for 3–5 days and then treated for 1–72 h with Al (AICI3) concentrations ranging from 10 to 75 μM at a constant pH of 4.1. In response to Al supply, lipid peroxidation in the Root Tips (< 2 cm) was enhanced only after longer durations of treatment. Aluminium-dependent increase in lipid peroxidation was intensified by Fe2+ (FeSO4). A close relationship existed between lipid peroxidation and inhibition of Root-elongation rate induced by Al and/or Fe toxicity and/or Ca deficiency. Besides enhancement of lipid peroxidation in the crude extracts of Root Tips due to Al, the activities of superoxide dismutase (EC 1.15.1.1) and peroxidase (EC 1.11.1.7) increased, whereas catalase (EC 1.11.1.6) activity decreased. This indicates a greater generation of oxygen free radicals and related tissue damage. The results suggest that lipid peroxidation is part of the overall expression of Al toxicity in Roots and that enhanced lipid peroxidation by oxygen free radicals is a consequence of primary effects of Al on membrane structure.
Cecelia Webster - One of the best experts on this subject based on the ideXlab platform.
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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
Plant Physiology, 2000Co-Authors: William W P Chang, Cecelia Webster, Lan Huang, Min Shen, Alma L Burlingame, Justin K M RobertsAbstract: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 [35S]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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contribution of malate and amino acid metabolism to cytoplasmic ph regulation in hypoxic maize Root Tips studied using nuclear magnetic resonance spectroscopy
Plant Physiology, 1992Co-Authors: Justin K M Roberts, Shaune Edwards, Mark A Hooks, Aaron P Miaullis, Cecelia WebsterAbstract:(31)P-, (13)C-, and (15)N-nuclear magnetic resonance spectroscopy were used to determine the roles of malate, succinate, Ala, Asp, Glu, Gln, and gamma-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.
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hypoxia enhances phosphorylation of eukaryotic initiation factor 4a in maize Root Tips
Journal of Biological Chemistry, 1991Co-Authors: Cecelia Webster, Rebecca L Gaut, Joanne M Ravel, Justin K M RobertsAbstract:We have identified two isoforms of initiation factor 4A (eIF-4A) in maize Root Tips, with distinct isoelectric points and similar molecular mass (approximately 50 kDa). Both isoforms of maize eIF-4A cross-react with antibodies raised against wheat germ eIF-4A, and one of the maize proteins (higher pI isoform) comigrates with purified wheat germ eIF-4A on two-dimensional gels. The two maize eIF-4As were indistinguishable by comparative peptide fingerprint analysis, which also showed a very strong similarity between eIF-4A in maize Roots and wheat germ. Maize eIF-4As copurify with eIF-4F and eIF-(iso)4F on a 7-methyl-GTP-Sepharose affinity column, indicating that they are part of the 5'-cap-binding complex. Two-dimensional gel electrophoresis and immunoblotting of proteins from 32P-labeled maize Root Tips revealed that the lower pI isoform of eIF-4A is phosphorylated. Two-dimensional phosphopeptide maps of trypsin-digested eIF-4A contained one principal phosphorylated fragment; phosphoamino acid analysis indicated phosphorylation of threonine. In oxygenated maize Root Tips, the ratio of phosphorylated to nonphosphorylated eIF-4A is approximately 0.2. This ratio increases to approximately 1 within 20 min following the onset of hypoxia, due to interconversion between the two maize eIF-4A isoforms. The hypoxia-induced phosphorylation of eIF-4A is discussed with respect to metabolic responses, and the translational control of gene expression, in hypoxic plant tissues.
