Ethanol attenuates lactate production in hypoxic postnatal day 4 rat cerebella.

Ethanol consumption during pregnancy may lead to a low oxygen supply to the brain of the developing fetus. Such a reduction in the oxygen supply will result in changes in intra- and extracellular lactate production, which subsequently may lead to cytoplasmic acidosis, changes in cerebral metabolism, and eventually, cell death. We used a novel application of gas chromatography to measure lactate changes, on a global level, in the cerebellar tissue of postnatal day (PD) 4 and PD 10 rat pups following in vitro exposure of either hypoxia or hypoxia plus ethanol (hypoxia/ethanol). The results showed hypoxia-induced increases in lactate concentrations as a function of treatment time in both PD 4 and PD 10 cerebellar tissue. However, there was a differential response to the additional ethanol treatment between the two age groups assessed, with an attenuation of the time-dependent increase of lactate production following hypoxia treatment in PD 4 cerebellar tissue. The results also indicated that PD 4 cerebellar tissue had increased oxygen utilization when compared with PD 10 tissue exposed to the same conditions. The ethanol-induced reduction in lactate is hypothesized as being due to limitations in glucose transport and utilization under ethanol/hypoxia exposure. It is believed that such limitations in cellular function may initiate a sequence of events that produce at least some of the cerebellar neuronal loss reported in the fetal alcohol literature.

A single mutation that increases maize seed weight.

The maize endosperm-specific gene shrunken2 (Sh2) encodes the large subunit of the heterotetrameric starch synthetic enzyme adenosine diphosphoglucose pyrophosphorylase (AGP; EC 2.7.7.27). Here we exploit an in vivo, site-specific mutagenesis system to create short insertion mutations in a region of the gene known to be involved in the allosteric regulation of AGP. The site-specific mutagen is the transposable element dissociation (Ds). Approximately one-third (8 of 23) of the germinal revertants sequenced restored the wild-type sequence, whereas the remaining revertants contained insertions of 3 or 6 bp. All revertants retained the original reading frame 3' to the insertion site and involved the addition of tyrosine and/or serine. Each insertion revertant reduced total AGP activity and the amount of the SH2 protein. The revertant containing additional tyrosine and serine residues increased seed weight 11-18% without increasing or decreasing the percentage of starch. Other insertion revertants lacking an additional serine reduced seed weight. Reduced sensitivity to phosphate, a long-known inhibitor of AGP, was found in the high seed-weight revertant. This alteration is likely universally important since insertion of tyrosine and serine in the potato large subunit of AGP at the comparable position and expression in Escherichia coli also led to a phosphate-insensitive enzyme. These results show that single gene mutations giving rise to increased seed weight, and therefore perhaps yield, are clearly possible in a plant with a long history of intensive and successful breeding efforts.

Identification and gene expression of anaerobically induced enolase in Echinochloa phyllopogon and Echinochloa crus-pavonis.

Enolase (2-phospho-D-glycerate hydrolase, EC 4.2.1.11) has been identified as an anaerobic stress protein in Echinochloa oryzoides based on the homology of its internal amino acid sequence with those of enolases from other organisms, by immunological reactivity, and induction of catalytic activity during anaerobic stress. Enolase activity was induced 5-fold in anoxically treated seedlings of three flood-tolerant species (E. oryzoides, Echinochloa phyllopogon, and rice [Oryza sativa L.]) but not in the flood-intolerant species (Echinochloa crus-pavonis). A 540-bp fragment of the enolase gene was amplified by polymerase chain reaction from cDNAs of E. phyllopogon and maize (Zea mays L.) and used to estimate the number of enolase genes and to study the expression of enolase transcripts in E. phyllopogon, E. crus-pavonis, and maize. Southern blot analysis indicated that only one enolase gene is present in either E. phyllopogon or E. crus-pavonis. Three patterns of enolase gene expression were observed in the three species studied. In E. phyllopogon, enolase induction at both the mRNA and enzyme activity levels was sustained at all times with a further induction after 48 h of anoxia. In contrast, enolase was induced in hypoxically treated maize root tips only at the mRNA level. In E. crus-pavonis, enolase mRNA and enzyme activity were induced during hypoxia, but activity was only transiently elevated. These results suggest that enolase expression in maize and E. crus-pavonis during anoxia are similarly regulated at the transcriptional level but differ in posttranslational regulation, whereas enolase is fully induced in E. phyllopogon during anaerobiosis.

Differential induction of mRNAs for the glycolytic and ethanolic fermentative pathways by hypoxia and anoxia in maize seedlings.

Fructose-1,6-biphosphate aldolase (ALD) and enolase (ENO) from the glycolytic pathway and pyruvate decarboxylase (PDC) and alcohol dehydrogenase 2 (ADH2) from the ethanolic fermentative pathway, are enzymes previously identified as among those synthesized selectively in O2-deficient roots of maize (Zea mays L.). The present study measured levels of transcripts representing these two pathways in 5-mm root tips, root axes (the remainder of the primary seminal root), and shoots of maize seedlings to determine how closely both pathways were co-induced and how they were modulated by changes in O2 concentration. In hypoxic seedlings with the roots in solution sparged with 5% (v/v) O2 (balance N2) and the shoots in the same gaseous atmosphere, mRNAs for Pdc1 and Adh2 in root tips both increased about 15-fold during the first 12 h, followed by a decline toward initial levels by 18 to 24h. Message levels for Ald1 and Eno1 showed only small changes during hypoxia. When expression was examined under anoxia, the extent to which all four mRNAs increased in different tissues depended on whether the seedlings had been previously acclimated to hypoxia or were anoxically shocked. The results show that although all the genes examined increased expression during hypoxia and/or anoxia, they differed in the rapidity and magnitude of the response and in the time to reach maximal message levels: there was no common pattern of change of message levels for the glycolytic or for the fermantative enzymes.

The Response of Maize Seedlings of Different Ages to Hypoxic and Anoxic Stress (Changes in Induction of Adh1 mRNA, ADH Activity, and Survival of Anoxia).

Previously we showed that there is only a transient induction of alcohol dehydrogenase 1 (Adh1) transcripts and only a small induction of alcohol dehydrogenase (ADH) enzyme activity in root tips of maize (Zea mays L.) seedlings subjected to strict anaerobiosis without prior acclimation by exposure to low O2 (D.L. Andrews, B.G. Cobb, J.R. Johnson, M.C. Drew [1993] Plant Physiol 101: 403-414). Acclimation of root tips of seedlings by low O2 before anoxia appeared to be necessary for full induction of ADH. Here we have examined the effect of seedling age on changes in the protein content, induction of Adh1 transcripts, and ADH enzyme activity in 5-mm root tips, root axes, and shoots of maize (cv TX5855). Their ability to survive anoxia was also recorded. Some seedlings were sparged with 4% O2 for 6 or 18 h (a hypoxic pretreatment) followed by anoxia (sparged with N2) for up to 48 h. Other seedlings were not acclimated before anoxia. In general, younger seedlings had higher initial (aerobic) levels of total protein, Adh1 transcripts, and ADH activity than did seedlings that were 2 d older. For younger seedlings, anoxia alone induced Adh1 transcripts, which reached a peak within 6 to 12 h, whereas ADH activity increased throughout the 48-h treatment. For older seedlings, anoxia caused only a small, transient induction of Adh1 transcripts or ADH activity. For seedlings of either age, hypoxia induced Adh1 transcripts and ADH activity, both of which were increased further by subsequent anoxia in the younger seedlings but to a lesser extent in the older seedlings. Despite differences in ADH activity, roots of seedlings of either age showed a similar resistance to anoxia. Thus, acclimation of maize seedlings to survive anoxia does not appear to be related to induction of high levels of ADH activity.

Hypoxic Induction of Anoxia Tolerance in Roots of Adh1 Null Zea mays L.

Seedlings of alcohol dehydrogenase 1 null mutants (Adh1-) of Zea mays L., which fail to synthesize alcohol dehydrogenase 1 (ADH1) isozymes, were hypoxically acclimated by 18 h of exposure to an atmosphere of 4% (v/v) O2 in N2 at 25[deg]C. Their ability to tolerate subsequent anoxia by exposure to anaerobic (O2-free) conditions was compared with that of unacclimated seedlings that were transferred immediately from an atmosphere of 40% (v/v) O2 to anaerobic conditions. Only 10% of the root tips of unacclimated seminal roots survived 6 h of anoxia, whereas 70% of the hypoxically acclimated root tips were viable at 24 h. During anoxia, acclimated root tips had enhanced ADH activity compared with unacclimated root tips, through induction of Adh2. Despite this, enzyme activity was still only about 5% that of acclimated, wild-type root tips and about half that of unacclimated, wild-type root tips. During anoxia, acclimated Adh1- root tips showed a higher rate of anaerobic respiration and ethanol production, greater concentrations of ATP and total adenylates, and a greater adenylate energy charge compared with unacclimated root tips. These results suggest that although enhanced ADH activity may have raised fermentation rates in acclimated Adh1- tissues and thereby contributed to energy metabolism and viability, the high levels of ADH activity inducible in acclimated, wild-type maize root tips appear to be in excess of that required to increase rates of fermentation.

Hypoxic and Anoxic Induction of Alcohol Dehydrogenase in Roots and Shoots of Seedlings of Zea mays (Adh Transcripts and Enzyme Activity).

Alcohol dehydrogenase (ADH) is one of a number of enzymes of glycolysis and fermentation known to be synthesized preferentially under low O2 conditions. We examined levels of Adh1 transcripts and of ADH activity in 5-mm root tips, root axes (the remainder of the seminal root), and shoots of maize (Zea mays L. cv TX 5855) seedlings. Seedlings with roots averaging about 60-mm long were transferred from fully aerobic conditions (solutions sparged with 40% [v/v] O2) to anaerobic (O2-free) conditions, or to an intermediate O2 concentration. There was no prior acclimation to low O2. In root tips, anoxia induced Adh1 transcripts and enzyme activity at 6 h, but this was followed by a rapid decline so that at 12 to 18 h neither were detectable and the root tips were dead. In contrast, higher levels of Adh1 transcripts and enzyme activity were maintained for at least 48 h in root axes and shoots. When induction at 6 h was measured over a wide range of O2 concentrations, a peak in ADH activity occurred in all tissues at 4% (v/v) O2. Maximum levels of transcripts, however, were in the range of 0 to 4% O2, depending on the tissue. The time course of hypoxic induction (at 4% O2) in root tips showed a peak in transcript levels at 6 h, whereas ADH activity continued to rise throughout the 24-h experiment. These results show that in root tips, ADH induction by anoxia was small and transient relative to induction by hypoxia.

Enhancement of Anaerobic Respiration in Root Tips of Zea mays following Low-Oxygen (Hypoxic) Acclimation.

Root tips (10-millimeter length) were excised from hypoxically pretreated (HPT, 4% [v/v] oxygen at 25 degrees C for 16 hours) or nonhypoxically pretreated (NHPT, 40% [v/v] oxygen) maize (Zea mays) plants, and their rates of respiration were compared by respirometry under aerobic and anaerobic conditions with exogenous glucose. The respiratory quotient under aerobic conditions with 50 millimolar glucose was approximately 1.0, which is consistent with glucose or other hexose sugars being utilized as the predominant carbon source in glycolysis. Under strictly anaerobic conditions (anoxia), glycolysis was accelerated appreciably in both HPT and NHPT root tips, but the rate of anaerobic respiration quickly declined in NHPT roots. [U-(14)C]Glucose supplied under anaerobic conditions was taken up and respired by HPT root tips up to five times more rapidly than by NHPT roots. When anaerobic ethanol production was measured with excised root tips in 50 millimolar glucose, HPT tissues consistently produced ethanol more rapidly than NHPT tissues. These data suggest that a period of low oxygen partial pressure is necessary to permit adequate acclimation of the root tip of maize to subsequent anoxia, resulting in more rapid rates of fermentation and generation of ATP.

Hypoxic Induction of Anoxia Tolerance in Root Tips of Zea mays.

When root tips of fully aerobic, intact maize (Zea mays L.) seedlings are made anaerobic, viability normally is only 24 hours or less at 25 degrees C. We find that viability can be extended to at least 96 hours if seedlings are given a hypoxic pretreatment for 18 hours by sparging the solution with 4% O(2) in nitrogen (v/v) before anoxia. Fully aerobic root tips (sparged with 40% O(2)) had very low alcohol dehydrogenase (ADH) activity (per gram root fresh weight), and the level remained low under anoxia. In hypoxically pretreated roots, however, high levels of ADH activity were induced, and activity rose further during the initial 24 hours of anoxia, and then remained high at about 20 times that of controls in 40% O(2). ADH activity in roots in solution sparged with air (21% O(2)) was about three times that in 40% O(2). Improved viability of hypoxically pretreated root tips was associated with maintenance of a high energy metabolism (ATP concentration, total adenylates, and adenylate energy charge). Roots that were not pretreated lost 94% of the total adenylates and ATP at 24 hours of anoxia. The relation between induced ADH activity, energy metabolism, and improved anoxia-tolerance in acclimated maize root tips is discussed.

Regulation of Embryo Dormancy by Manipulation of Abscisic Acid in Kernels and Associated Cob Tissue of Zea mays L. Cultured in Vitro.

Sectors of Zea mays cobs, with and without kernels were cultured in vitro in the presence and absence of fluridone. Cultured kernels, cob tissue, and embryos developed similarly to those grown in the field. Abscisic acid (ABA) levels in the embryos were evaluated by enzyme-linked immunosorbant assay. ABA levels in intact embryos cultured in the presence of fluridone were extremely low and indicate an inhibition of ABA synthesis. ABA levels in isolated cob tissue indicate that ABA can be produced by cob tissue. Sections containing kernels cultured in the presence of fluridone were transferred to medium containing fluridone and ABA. Dormancy was induced in more than 50% of the kernels transferred from 13 to 15 days after pollination, but all of the kernels transferred at 16 days after pollination or later were viviparous. ABA recovered from kernels that were placed in medium containing fluridone and ABA suggest that ABA can be transported through the cob tissue into developing embryos and that ABA is required for induction of dormancy in intact embryos.

Vitrification and soluble carbohydrate levels inPetunia leaves as influenced by media gelrite and sucrose concentrations.

Normal nodal segments ofPetunia hybrida were grown on Murashige and Skoog salts containing varied levels of Gelrite and sucrose. Higher concentrations of Gelrite decreased vitrification while increased sucrose concentrations promoted vitrification. Leaves of vitreous plants had higher levels of reducing sugars and sucrose but lower or undetectable levels of inositol as compared to normal plants. Normal plants on medium void of inositol have the ability to synthesize inositol and maintain levels equal to that found in plants from inositol containing media.

Shrunken-1 encoded sucrose synthase is not required for sucrose synthesis in the maize endosperm.

Kernels of wild-type maize (Zea mays L.) shrunken-1 (sh1), deficient in the predominant form of endosperm sucrose synthase and shrunken-2 (sh2), deficient in 95% of the endosperm ADP-glucose pyrophosphorylase were grown in culture on sucrose, glucose, or fructose as the carbon source. Analysis of the endosperm extracts by gas-liquid chromatography revealed that sucrose was present in the endosperms of all genotypes, regardless of carbon supply, indicating that all three genotypes are capable of synthesizing sucrose from reducing sugars. The finding that sucrose was present in sh1 kernels grown on reducing sugars is evidence that shrunken-1 encoded sucrose synthase is not necessary for sucrose synthesis. Shrunken-1 kernels developed to maturity and produced viable seeds on all carbon sources, but unlike wild-type and sh2 kernels grown in vitro, sucrose was not the superior carbon source. This latter result provides further evidence that the role of sucrose synthase in maize endosperm is primarily that of sucrose degradation.

Protein synthesis in slash pine callus cultures exposed to water stress.

Effects of water stress on protein synthesis were investigated in heterotrophic callus cultures derived from slash pine (Pinus elliottii Engelm.) cotyledon explants. Cultures were transferred to medium containing 0-15% mannitol to provide water potentials between -0.4 and -2.5 MPa. Beginning between 0 and 22 h after transfer to the new medium, cultures were incubated for 2 h with (35)S-methionine. Both the uptake of label and the incorporation of label into protein decreased with decreasing medium water potential. Incorporation, however, was reduced to a greater extent than uptake suggesting that the reduced incorporation was at least partly the result of reduced protein synthesis. Separation of labeled proteins by one-dimensional SDS-polyacrylamide gel electrophoresis and visualization by fluorography revealed the induction of two new protein bands after incubation at -1.8 MPa for 24 h. Induction of new proteins was not observed at other water potentials or after shorter incubation times. Protein bands from cultures incubated at -1.8 MPa for 24 h and then transferred for 24 h to mannitol-free medium (-0.4 MPa) were comparable to those from control cultures maintained on mannitol-free medium throughout.

Sugar utilization by developing wild type and shrunken-2 maize kernels.

To characterize the movement of sugars during kernel development in maize, a newly devised in vitro kernel development scheme was utilized. Viable seeds of wild type maize (Zea mays L.) as well as the mutant shrunken-2 (sh2) were found to mature when grown in culture with reducing sugars or sucrose as the carbon source. However, wild type and sh2 kernels had greater germination, starch content, and seed weight when sucrose, rather than reducing sugars, was the carbon source. By the use of labeled sucrose it was shown that sucrose can move into endosperm tissue without intervening degradation and resynthesis. These results show that when grown in vitro the maize seed can utilize reducing sugars for development, but it prefers sucrose.

Development of wild type, shrunken-1 and shrunken-2 maize kernels grown in vitro.

Kernels of wild type maize (Zea mays L.) and the mutants shrunken-1 and shrunken-2 developed as much as in vivo when excised at five days post-pollination and grown in culture using existing methods. Mature kernels from culture exhibited their expected phenotypes. Starch, sugar and enzyme levels of kernels grown in culture were similar to those known to occur in kernels of the same genotypes grown in vivo. Differences in percentage germination of kernels grown in vitro were similar to those of kernels grown in vivo.