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°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% O₂ in nitrogen (v/v) before anoxia. Fully aerobic root tips (sparged with 40% O₂) 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% O₂. ADH activity in roots in solution sparged with air (21% O₂) was about three times that in 40% O₂. 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.     

Metabolic Acclimation to Anoxia Induced by Low (2-4 kPa Partial Pressure) Oxygen Pretreatment (Hypoxia) in Root Tips of Zea mays

Young intact plants of maize (Zea mays L. cv INRA 508) were exposed to 2 to 4 kilopascals partial pressure oxygen (hypoxic pretreatment) for 18 hours before excision of the 5 millimeter root apex and treatment with strictly anaerobic conditions (anoxia). Hypoxic acclimation gave rise to larger amounts of ATP, to larger ATP/ADP and adenylate energy charge ratios, and to higher rates of ethanol production when excised root tips were subsequently made anaerobic, compared with root tips transferred directly from aerobic to anaerobic media. Improved energy metabolism following hypoxic pretreatment was associated with increased activity of alcohol dehydrogenase (ADH), and induction of ADH-2 isozymes. Roots of Adh1⁻ mutant plants lacked constitutive ADH and only slowly produced ethanol when made anaerobic. Those that were hypoxically pretreated acclimated to anoxia with induction of ADH2 and a higher energy metabolism, and a rate of ethanol production comparable to that of nonmutants. All these responses were insensitive to the presence or absence of NO₃⁻. Additionally, the rate of ethanol production was about 50 times greater than the rate of reduction of NO₃⁻ to NO₂⁻. These results indicate that nitrate reductase does not compete effectively with ADH for NADH, or contribute to energy metabolism during anaerobic respiration in this tissue through nitrate reduction. Unacclimated root tips of wild type and Adhl⁻ mutants appeared not to survive more than 8 to 9 hours in strict anoxia; when hypoxically pretreated they tolerated periods under anoxia in excess of 22 hours.     

Aluminum Partitioning in Intact Roots of Aluminum-Tolerant and Aluminum-Sensitive Wheat (Triticum aestivum L.) Cultivars

Aluminum (Al) partitioning in intact roots of wheat (Triticum aestivum L.) cultivars that differ in sensitivity to Al was investigated. Roots of intact seedlings were exposed to Al for up to 24 hours and distribution of Al was assessed visually by hematoxylin staining or by direct measurement of concentration of Al by atomic absorption spectrophotometry or ion chromatography. Major differences in Al accumulation between Al-tolerant (Atlas 66) and Al-sensitive (Tam 105) cultivars were found in the growing regions 0 to 2 and 2 to 5 millimeters from the root apex. Al content was 9 to 13 times greater in the 0 to 2 millimeters root tips of cv Tam 105 than in the tips of cv Atlas 66 when exposed to 50 micromolar Al for 19 to 24 hours. The oxidative phosphorylation inhibitor carbonyl cyanide m-chlorophenylhydrazone and the protein synthesis inhibitor cycloheximide increased Al uptake by intact root tips of cv Atlas 66. Also, loss of Al from the roots of both cultivars was measured after the roots were “pulsed” with 50 micromolar Al for 2 hours and then placed in an Al-free nutrient solution for 6 hours. The 0 to 2 millimeter root tips of cv Tam 105 lost 30% of the absorbed Al, whereas the tips of cv Atlas 66 lost 60%. In light of these results, we conclude that the differential Al sensitivity in wheat correlates with the concentration of Al in the root meristems. The data support the hypothesis that part of the mechanism for Al tolerance in wheat is based on a metabolism-dependent exclusion of Al from the sensitive meristems.     

Quantitative Localization of the Phytoalexin Glyceollin I in Relation to Fungal Hyphae in Soybean Roots Infected with Phytophthora megasperma f. sp. glycinea

A radioimmunoassay specific for glyceollin I was used to quantitate this phytoalexin in roots of soybean (Glycine max [L.] Merr. cv Harosoy 63) after infection with zoospores of either race 1 (incompatible) or race 3 (compatible) of Phytophthora megasperma Drechs. f. sp. glycinea Kuan and Erwin. The sensitivity of the radioimmunoassay and an inmmunofluorescent stain for hyphae permitted quantitation of phytoalexin and localization of the fungus in alternate serial cryotome sections from the same root. The incompatible interaction was characterized by extensive fungal colonization of the root cortex which was limited to the immediate vicinity of the inoculation site. Glyceollin I was first detected in extracts of whole roots 2 hours after infection, and phytoalexin content rose rapidly thereafter. Significant concentrations of glyceollin I were present at the infection site in cross-sections (42 micrometers thick) of such roots by 5 hours, and exceeded 0.6 micromoles per milliliter (EC₉₀in vitro for glyceollin I) by 8 hours after infection. Longitudinal sectioning (14 micrometers thick) showed that glyceollin I accumulated particularly in the epidermal cell layers, but also was present in the root cortex at inhibitory concentrations. No hyphae were observed in advance of detectable levels of the phytoalexin and, in most roots, glyceollin I concentrations dropped sharply at the leading edge of the infection. In contrast, the compatible interaction was characterized by extensive unchecked fungal colonization of the root stele, with lesser growth in the rest of the root. Only small amounts of glyceollin I were detected in whole root extracts during the first 14 hours after infection. Measurable amounts of glyceollin I were detected only in occasional cross-sections of such roots 11 and 14 hours after infection. The phytoalexin was present at inhibitory concentrations in the epidermal cell layers, but the inhibitory zone did not extend appreciably into the cortex. Altogether, these data support the hypothesis that the accumulation of glyceollin I is an important early response of soybean roots to infection by P. megasperma, but may not be solely responsible for inhibition of fungal growth in the resistant response.     

Inhibitory action of auxin on root elongation not mediated by ethylene

The inhibitory effects of indole-3-acetic acid (IAA) and 1-aminocyclopropane-1-carboxylic acid (ACC) on elongation growth of pea (Pisum sativum L.) seedling roots were investigated in relation to the effects of these compounds on ethylene production by the root tips. When added to the growth solution both compounds caused a progressively increasing inhibition of growth within the concentration range of 0.01 to 1 micromolar. However, only ACC increased ethylene production in root tips excised from the treated seedlings after 24 hours. High auxin concentrations caused a transitory increase of ethylene production during a few hours in the beginning of the treatment period, but even in 1 micromolar IAA this increase was too low to have any appreciable effect on growth. ACC, but not IAA, caused growth curvatures, typical of ethylene treatment, in the root tips. IAA caused conspicuous swelling of the root tips while ACC did not. Cobalt and silver ions reversed the growth inhibitory effects induced by ACC but did not counteract the inhibition of elongation or swelling caused by IAA. The growth effects caused by the ACC treatments were obviously due to ethylene production. We found no evidence to indicate that the growth inhibition or swelling caused by IAA is mediated by ethylene. It is concluded that the inhibitory action of IAA on root growth is caused by this auxin per se.     

Effect of aboveground litter on belowground plant interactions in a native Rough Fescue grassland

Chemical compounds from plants may exhibit stimulatory and/or inhibitory effects on surrounding organisms. However, research on belowground biochemical interactions among plants has focused more effort on elucidating negative effects. Moreover, the effect of shoot litter on belowground plant–plant interactions has remained relatively unexplored. In a field experiment with four target plant species (Artemisia frigida Willd., Solidago missouriensis Nutt.), Bouteloua gracilis (Willd. ex Kunth) Lag. ex Griffiths and Poa pratensis L.) interacting with intact grassland neighbours, we manipulated root competition using PVC tubes and shoot litter, and belowground chemical interaction by adding activated carbon (AC) to the soil. In A. frigida, shoot litter significantly interacted with root competition and root chemicals. Plants grown plus AC were larger than those minus AC when shoot litter was left intact suggesting inhibitory effects from neighbours and/or decomposing products. However, when shoot litter was removed, plants grown minus AC were larger suggesting stimulatory effects of root exudates. B. gracilis showed a similar trend but results were non-significant. Results demonstrate that the effects of neighbours can be inhibitory or facilitative depending on the presence or absence of shoot litter and mediation through AC.     

Alternative Path Mediated ATP Synthesis in Roots of Pisum sativum upon Nitrogen Supply

Changes in the efficiency of root respiration were examined on intact plants of Pisum sativum L. cv Rondo after addition of nitrate or ammonium to the culture solutions. Nitrate was absorbed immediately after addition and elicited a respiratory rise (O₂-uptake as well as CO₂-production) to 160% at most. This occurred both in roots of plants fixing N₂ and in those of non-nodulated plants pregrown for 1 or 2 weeks on a nitrogen-free culture solution. In older plants, used after 2 weeks of N-free growth, the full capacity of the cytochrome path was engaged in root respiration. This was demonstrated by the absence of an effect of the uncoupler carbonylcyanide m-chlorophenylhydrazone in the presence of 25 millimolar salicylhydroxamate, an inhibitor of the alternative path. In these plants more than 90% of the nitrate-induced stimulation of root respiration was salicylhydroxamate-sensitive. In young plants, used after 1 week of N-free growth, the cytochrome path was not saturated. Its activity increased instantaneously at the expense of alternative path activity, which initially dropped to zero and subsequently increased to 160% of the control 7 hours after nitrate supply. The rate of photosynthesis rose to 120% of the control, but not before 1 hour after nitrate supply, suggesting that the stimulation of root respiration was not due to a higher rate of photosynthesis. Experiments with plants grown with a split-root system showed that respiration rate and alternative path activity only increased in the root halves exposed to nitrogen. Ammonium was equally effective as nitrate in stimulating root respiration. These results lead to the conclusion that alternative-path mediated root respiration contributes to synthesis of ATP during at least the first 24 hours following nitrogen supply.     

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°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-¹⁴C]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.     

Plant-Induced Hatching of Eggs of the Soybean Cyst Nematode Heterodera glycines

Root diffusate from soybean plants caused greater hatching of Heterodera glycines eggs during vegetative growth of the host, but the activity declined with plant senescence. Chelation of the root diffusate with ethylenediamine tetraacetic acid (EDTA) significantly increased hatching activity for H. glycines eggs. Diffusate from leafless plants caused little hatching, whereas treatment of intact plants with the growth regulators gibberellin and kinetin had no effect on the hatching activity of root diffusate. Treating H. glycines eggs with zinc chloride and root diffusate reduced egg hatching from zinc chloride alone. Levels of zinc in the root diffusate were insufficient to induce egg hatch, based on analysis by atomic absorption spectrophotometry. The enzymatic activity of leucine aminopeptidase in H. glycines eggs was not altered by treatment with chelated or nonchelated root diffusate.     

Rubidium Absorption by Corn Root Tissue after a Brief Period of Water Stress and during Recovery

Intact and excised samples of corn root tissue were subjected to water stress either by incubation in solutions of osmotica or by desiccation, after which they were transferred to CaSO₄ solution for various time periods for recovery. Osmotic agents used were either mannitol or polyethylene glycol 6000 at concentrations adequate to depress rubidium absorption to less than 30% of that of controls. During 6 h following release from osmotic stress, rubidium absorption by samples from intact seedlings treated with mannitol increased to 44% of that of controls, while those treated with polyethylene glycol increased to 79% of that of controls. Recovery of root samples excised prior to stress was very nearly the same as that of samples from intact roots. When water stress was produced by desiccation, recovery was about the same as from polyethylene glycol, attaining a rate of 76% of that of controls after 8 h. Whereas desiccated samples almost completely regained their water content during recovery, none showed correspondingly complete recovery of ion absorption capacity. Finally, during the recovery period, the submerged controls, but not controls in humid air, showed an increase in ion absorption capacity with time, confirming that the widely observed “aging effect” or “washing effect” is due to submersion and not to time per se.     

Effect of removal of the root tip on the development of enhanced rb absorption by corn roots

Samples of primary root tissue of corn (Zea mays L.) were aged either in CaSO(4) solution or in humid air, after which they were immersed for 10 minutes in a solution containing 0.1 mm(86)RbCl. Aging in solution, but not in humid air, enhanced the subsequent rate of Rb(+) absorption. Excision of roots before aging was followed by greater enhancement than when exicision followed aging. The time course of aging of 1-cm segments from different portions of the root showed decreasing response with increasing distance from the root cap. The aging response of apical segments (5-15 mm from the root cap) could be detected within 10 minutes and usually reached a maximum within 2 hours. Rb(+) absorption by apical segments (5-15 mm) aged without the tip (0-5 mm) was more than double that by apical segments whose tips were left attached until the end of the aging period. When apical segments without the tip were aged for 2 hours in the CaSO(4) solution in which seedlings had previously been grown for 24 hours, the rate of absorption was only 63% of samples aged in fresh solution. When apical segments were aged for 2 hours in fresh solution containing excised tips floating free in the solution, the rate of Rb(+) absorption was 20% less than in samples aged in solution containing no excised tips. The data presented in this study are interpreted to indicate that a water-soluble metabolite, originating in the root tip and translocated basipetally, inhibits Rb accumulation.     

The kinetics of carotenoid absorption changes in intact cells of photosynthetic bacteria

The kinetics of carotenoid absorption changes have been measured in intact cells of Rhodopseudomonas capsulata after short flash excitation. The observed changes were consistent with the thesis that they indicate the development and dissipation of membrane potential. In the generation of the absorption changes in anaerobic cells, fast (complete in 0.5 ms) and slow (half-time 3 ms) components can be distinguished. The slow component corresponds kinetically to the rate of cytochrome c re-reduction and is similarly antimycin sensitive. These data are similar to those observed in isolated chromatophores which have been artifically poised with redox mediators. In aerobic intact cells the kinetic profile is altered, mainly because the decay of the carotenoid change is much faster. Inhibition of respiration with KCN leads to flash-induced changes similar to those in anaerobic cells. At least two components can be distinguished in the decay of the carotenoid absorption changes in anaerobic intact cells. Only the faster decay component was inhibited by venturicidin which suggests that it corresponds to H⁺ flux through the F₀F₁-ATPase during ATP synthesis. The contribution of the venturicidin-sensitive decay to the total decay was dependent upon the initial amplitude of the carotenoid absorption change produced by the flash group. This suggests that there is an apparent threshold of membrane potential for ATP synthesis. Supporting evidence was provided by the finding that venturicidin stimulated the steady-state light-induced carotenoid absorption change at high but not at low light intensities. The entire decay of the carotenoid absorption changes was stimulated by carbonyl cyanide p-trifluoromethoxyphenylhydrazone in a manner that can be interpreted as an ionophore catalysing the dissipation of membrane potential.     

Greening of etiolated bean leaves in far red light

Eight-day-old dark-grown bean leaves were greened by prolonged irradiation with far red light. Growth, chlorophyll content, oxygen-evolving capacity, photophosphorylation capacity, chloroplast structure (by electron microscopy), and in vivo forms of chlorophyll (by low temperature absorption and derivative spectroscopy on intact leaves) were followed during the greening process. Chlorophyll a accumulated slowly but continuously during the 7 days of the experiment (each day consisted of 12 hours of far red light and 12 hours of darkness). Chlorophyll b was not detected until the 5th day. The capacity for oxygen evolution and photophosphorylation began at about the 2nd day. Electron microscopy showed little formation of grana during the 7 days but rather unfused stacks of primary thylakoids. The thylakoids would fuse to give grana if the leaves were placed subsequently in white light. The low temperature spectroscopy of intact leaves showed that the chlorophyll a was differentiated into three forms with absorption maxima near 670, 677, and 683 nanometers at −196 C during the first few hours and that these forms accumulated throughout the greening process. Small amounts of two longer wavelength forms with maxima near 690 and 698 nanometers appeared at about the same time as photosynthetic activity.     

Effect of boron on cell elongation and division in squash roots

This work establishes that cessation of root elongation of intact squash (Cucurbita pepo L.) plants is an early result of boron deficiency. Root elongation is slowed by 6 hours and is virtually stopped as early as 24 hours after boron is first withheld from the nutrient solution. As root elongation ceased, cell elongation progressed distally into the region normally occupied by the apical meristem and eventually the meristem became indistinguishable. Differentiation was determined by use of an elongation index in which cell length was compared to cell width. This index ranged from a low of 0.8 in boron-sufficient root meristems to a high of 3 in root meristems grown in a boron-deficient nutrient solution for 98 hours. It is concluded that a continuous supply of boron is not essential for cell elongation but is required for maintenance of meristematic activity. Boron may act as a regulator of cell division in this tissue.     

Intervention de l'oxygène atmosphériqoe sur l'absorption d'eau chez Helianthus annuus

The role of atmospheric oxygen on root water absorption in Helianthus annuus . The effect of atmospheric anoxia on root water absorption was studied. The experiments were carried out on intact young sunflowers in controlled temperature, light and gas environment; roots were kept in aerated nutrient solution at constant temperature. The evolution of root water absorption and transpiration rate was measured continuously. Before the experiment, the plant was preconditioned at a high transpiration rate by illumination or by CO₂ free air in darkness. Then the atmospheric oxygen was suppressed for 1 h, after which the normal conditions were restored.
In anoxia and darkness, the root water absorption cannot balance transpiration, so that an important water stress develops in the plant; the light compensates this effect through the photosynthetic oxygen. The supply of oxygen, in darkness or in light, immediately removes inhibition of stomatal closure and of root water absorption. Two mechanisms control water absorption by roots: the fast one occurs in the leaves and the slower one cannot develop without the root system.     

Increased electron density of tonoplast membranes in washed corn root tissue

Washing corn (Zea mays L.) root segments for 4 hours results in increased tonoplast electron density in fixed and stained tissue. It suggested that previously reported increases in solute absorption rate with root tissue washing may lie with changes in the properties of the tonoplast.     

Deoxyribonucleic Acid synthesis in root cap cells of cultured roots of convolvulus

Isolated cultured roots of Convolvulus arvensis L. were incubated in 0.2 microcurie per milliliter methyl-³H-thymidine for 14 hours, for 64 hours, or for 14 hours followed by transfer to fresh nutrient medium without tritiated thymidine. Autoradiographs of serial, longitudinal sections of roots which were continuously incubated with tritiated thymidine showed that cells of the root cap columella did not undergo DNA synthesis after their formation from the root cap initials. In roots pulse-labeled with tritiated thymidine, the movement of labeled cells through the root cap columella was followed. Labeled cells were displaced at a constant rate of 72 microns per day over a period of 6 to 9 days before they were sloughed off from the root cap. The specialized role of the root cap cells in relation to their distinctive metabolism and longevity is discussed.     

Inhibitory action of estradiol on growth promoting activity in extract from uterine cancers

Extracts from uterine cervical and body cancers, but not from benign tumor or intact tissues tested, were found to contain a growth-promoting activity which induced the proliferation of human endometrial fibroblasts. Exposure of cultured fibroblasts to the cancer extracts increased the rate of [³H]thymidine incorporation in a dose-dependent manner. The activity was heat-labile, and not inactivated by removal of lipid-soluble material suggesting that the activity is associated with a protein. When the fibroblasts were preincubated with estradiol for 12 hours, but not for 1 hour, the extract-induced fibroblast proliferation was suppressed. The inhibitory effect of estradiol was dose related (EC₅₀: 10 nM) and non-competitive, suggesting that the steroid may reduce the sensitivity of fibroblasts to the extracts. This is the first report to provide direct evidence that estradiol may play an inhibitory role in the action of growth factor-like peptide produced from malignant tumors.     

Population Dynamics,Root Penetration,and Feeding Behavior of Pratylenchus agilis in Monoxenic Root Cultures of Corn,Tomato, and Soybean

Population dynamics, rate of root penetration, and external root feeding behavior of Pratylenchus agilis (Pa) in monoxenic cultures of intact corn seedlings and root explants of corn, tomato, and soybean were studied. In descending order of suitability as hosts were I. O. Chief corn, Rutgers tomato, and Williams soybean. Soybean entries Kent, Pickett 71, PI 90763, and Essex were poor hosts. Numbers of eggs and vermiform Pa in the agar medium indicated total fecundity and host suitability. Agar, sand, or soil as support media did not appear to affect Pa root penetration, but the rate of corn root growth did. Whereas most vermiform Pa and eggs were in roots, substantial numbers appeared able to feed and complete their life cycle as ectoparasites on root epidermal cells and root hairs.     

Effect of Soybean Tip Removal on Penetration and Development of Heterodera glycines

On a few occasions, soybeans with broken root tips were included in tests to evaluate resistance to Heterodera glycines. Although females developed on these plants, the numbers tended to be lower than on similarly treated intact roots. To test the possibility that removal of the root meristem affected nematode development, a culture system using pruned soybeans was devised that permitted access to the roots without disturbing the plants. Treatments included removal of 2 mm of root tip at various times ranging from 24 hours before to 10 days after inoculation, or roots left intact. In each experiment, all roots were inoculated at the same time with equal numbers of freshly hatched second-stage juveniles of Heterodera glycines. No differences in nematode development were detected in plants with root tips removed after inoculation compared to the control. When tips were removed at or before inoculation, fewer juveniles entered roots and relatively fewer nematodes developed. Penetration levels and development correlated with root tip removal such that progressively fewer nematodes entered roots and relatively greater numbers of nematodes remained undeveloped as the time interval between root tip removal and inoculation was increased.