Localization of Acid Hydrolase Activity in Zea mays L. Root Tips

Naphthol AS-BI phosphatase, esterase, aryl sulphatase, glucuronidase,and ß-glycerophosphatase have been studied in frozensections of maize root tips. In general these enzymes showedhighest activities at the root surface and at particulate sitesin the cytoplasm although the indigogenic method for esteraseshowed no particulate activity and the naphthol AS-D acetatereaction gave no pronounced surface activity. With electronmicroscopy highest activity for ß-glycerophosphatasewas observed in the cell walls and associated with the vacuoles.The significance of these observations are discussed in relationto the function of surface hydrolytic activity and to the presenceof lysosome-like bodies in higher plant cells.     

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.     

Longitudinal Water Movement in the Primary Root of Zea mays

The rates of transfer of tritiated water (THO) along lengthsof excised primary roots of Zea mays have been measured undera variety of conditions. The following values of ‘apparentdiffusion coefficients’ for THO in the root tissue havebeen evaluated: 1.5±0.1x10^-5 cm² sec^-1 in roots boiledfor 3 min before use,0.5±0.03x10^-5 cm² sec^-1 in rootspoisoned with 10^-2 M NaF,0.9±0.07x10^-5 cm² sec^-1 in rootspoisoned with 10^-2 M NaN₃,and 2.1±0.2x10^-5 cm² sec^-1in normal roots. The bathing medium in all cases was 1.0 mMKCl/0.1 mM CaCl₂ with the addition of the inhibitors where appropriate.Thefourfold increase in the rate of THO transfer in normal rootscompared with poisoned ones is attributed to the existence ofa long-distance convective flow in the first case, which isterminated by the addition of inhibitors. Since experimentsshow that this convective flow must occur both acropetally andbasipetally with equal velocity, it is thought to occur in thephloem.By assuming the ‘streaming transcellular strands’model for phloem transport, the rate of movement required togive the observed transfer has been computed as approximately4.5x10^-2 cm sec^-1 (160 cm h^-1).The earlier report of the existenceof a highly impermeable barrier surrounding the xylem vesselshas been further substantiated by the experiments reported here.     

Calcium-Regulated in Vivo Protein Phosphorylation in Zea mays L. Root Tips

Calcium dependent protein phosphorylation was studied in corn (Zea mays L.) root tips. Prior to in vivo protein phosphorylation experiments, the effect of calcium, ethyleneglycol-bis-(β-aminoethyl ether)-N-N′-tetraacetic acid (EGTA) and calcium ionophore (A-23187) on phosphorus uptake was studied. Calcium increased phosphorus uptake, whereas EGTA and A-23187 decreased it. Consequently, phosphorus concentration in the media was adjusted so as to attain similar uptake in different treatments. Phosphoproteins were analyzed by two-dimensional gel electrophoresis. Distinct changes in phosphorylation were observed following altered calcium levels. Calcium depletion in root tips with EGTA and A-23187 decreased protein phosphorylation. However, replenishment of calcium following EGTA and ionophore pretreatment enhanced phosphorylation of proteins. Preloading of the root tips with ³²P in the presence of EGTA and A-23187 followed by a ten minute calcium treatment, resulted in increased phosphorylation indicating the involvement of calcium, calcium and calmodulin-dependent protein kinases. Calmodulin antagonist W-7 was effective in inhibiting calcium-promoted phosphorylation. These studies suggest a physiological role for calcium-dependent phosphorylation in calcium-mediated processes in plants.     

Correlation between Root-Generated Ionic Currents, pH, Fusicoccin, Indoleacetic Acid, and Growth of the Primary Root of Zea mays

Correlations between root-generated ionic currents, extracellular pH, indoleacetic acid, fusicoccin, and growth were investigated. Current consistently entered the meristematic and elongating tissues of intact growing roots of Zea mays cv Golden Bantam. Mature root regions generated the outward limb of the current loop. Ion-substitution and pH-profile experiments suggested that the bulk of the ionic current was carried by H⁺. Calcium ions did not carry current, but calcium may regulate the proton circulation since the proton current density was slightly larger in calcium-depleted media. Increased root elongation at low pH was associated with increased current density and an extended zone of inward current. Conversely decreased elongation at high pH was associated with a reduced current density and a more restricted zone of inward current. The effect of the fungal toxin fusicoccin was to increase the current density of the inward limb of the ion current and to increase root extension. Concentrations of indoleacetic acid that reduced root growth, also reduced the density of the inward current and shortened the inward current zone. The results emphasize the point that roots are electrically contiguous over many millimeters and that the electrophysiology of root growth is best studied in intact root systems.     

Indoleacetic Acid and Abscisic Acid Antagonism: II. On the Phytochrome-Mediated Attachment of Barley Root Tips on Glass

The phytochrome-mediated attachment of mung bean (Phaseolus vulgaris L., var. Oklahoma 612) root tips on glass is quickly affected by indoleactic acid and abscisic acid at concentrations of 10 nm or less. Indoleacetic acid induces detachment, whereas abscisic acid induces attachment. Both plant regulators rapidly antagonize the action of the other. None of several cytokinins, gibberellins, and ethylene tested over a wide range in concentration had any effect on either attachment or detachment of root tips. It is postulated that phytochrome could control the endogenous levels of indoleacetic acid and abscisic acid and perhaps other hormones under certain circumstances, that this action is the first process initiated by phytochrome, and that indoleacetic acid and abscisic acid act on the plasmalemma to bring about opposing changes in the surface electric charges of plant cells.     

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.     

Salt Stress Causes Acceleration of Purine Catabolism and Inhibition of Pyrimidine Salvage in Zea mays Root Tips

Nucleotide metabolism was studied in apical 5.0 mm root tipsof corn plants (Zea mays L., cv. Pioneer 3906) hydroponicallycultured for 7 d and then salinized for 19 d at a rate calculatedto reduce the osmotic potential (_o) of the solutions by O.1MPad^–1 to a final _o = -0.4 MPa. Saline treatments withtwo different molar ratios of Ca₂₊/Na⁺ were employed, viz.,0–03 (2.5 mol m^–3 CaCl₂ + 86.5 mol m^–3 NaCl)for the NaCl treatment and 0.73 (31.5 mol m^–3 CaCl₂ +43.1 mol m^–3 NaCl) for the NaCl + CaCl₂ treatment. Bothsalt treatments reduced root growth by more than 30%. The capacityof roots to provide purine nucleotides either by de novo synthesisor by re-utilization of existing bases, e.g. salvage of hypoxanthineto adenine nucleotides, was not affected by either salt treatment.However, catabolism of hypoxanthine was accelerated more than3.5-fold by both salt treatments, demonstrating an increasedcapacity for purine catabolism which would shift the normal1: 1 ratio of synthesis: degradation of purine nucleotides observedfor the roots of healthy control plants to less than 0.2 duringsalt stress. The ratio of pyrimidine nucleotide synthesis: degradationwas also reduced. In this case, the unfavourable shift towardnucleotide degradation resulted because both salt treatmentsreduced salvage capacity by more than 25%, but had no compensatingeffect on de novo synthesis or catabolism of pyrimidines. Key words: Salinity, osmotic potential, nucleotide metabolism     

The Effects of Mechanical Impedance to Growth on the Levels of ABA and IAA in Root Tips of Zea mays L.

Extraction and analytical methods have been refined and newones devised to allow precise determinations by GC-EC of thelevels of abscisic acid (ABA) and indol-3ylacetic acid (IAA)in samples of maize root tips as small as 1.0 g fr. wt. Seminalroots of 5-d-old maize seedlings grown in normal (bulk density1200 kg m^–3) and compacted (bulk density 1600 kg m^–3)sand/garden loam mixtures have been examined. Seminal rootsfrom compacted soil had an average length of about 40% of thatof control roots and were much thicker. The ABA levels in 10mm tips of impeded roots (c. 25–35 ng g^–1 fr.wt.)did not differ significantly from those of normal root tipson both a fresh and dry weight basis. The levels in 0–1mm tips were approximately double those in the remaining 1–10mm zones. IAA levels were increased by about 3 times in impededroots (176.3 as compared with 52.4 ng g^–1 fr.wt) and itis concluded that this response is likely to be the main causeof the morphological and growth changes brought about by soilcompaction.     

Nucleotide Metabolism in Salt-Stressed Zea mays L. Root Tips: I. Adenine and Uridine Nucleotides

Corn plants (Zea mays L. cv Pioneer 3906) were grown in a glass house on control and saline nutrient solutions, in winter and summer. There were two saline treatments, both with osmotic potential = −0.4 megapascal but with different Ca²⁺/Na⁺ ratios: 0.03 and 0.73. Root tips and shoot meristems (culm tissue) of 26 day-old plants were analyzed for nucleotides to ascertain if there were correlations between nucleotide pool size and the reduced growth on saline cultures. Several other cell components also were determined. Plants grown in winter were only half as large as those grown in summer mainly because of the lower light intensity and lower temperature. But the relative yield reduction on salt treatment compared to the control was similar in winter and summer. The two different salt treatments caused similar yield reductions. Neither salt treatment affected nucleotide pools in culm tissue, with the possible exception of UDPG in winter. In the case of root tips, salt treatment had little or no effect on nucleotide pool sizes in winter when many already seemed near a critical minimum, but in summer it reduced several pools including ATP, total adenine nucleotide, UTP, total uridine nucleotide, and UDP-glucose. The reductions were greatest on the salt treatment with low Ca²⁺/Na⁺. There was no simple correlation between the effects of salt stress on growth and on nucleotide pool size. The nucleotide pools of culm tissue indicated that in some respects this tissue was effectively insulated from the salt stress. Roots that were in direct contact with the saline solution indicated significant reductions in nucleotide pools only in the summer whereas growth was reduced both summer and winter. It is possible that the nucleotide concentrations of root cells in winter were already near a critical minimum so that nucleotide synthesis and growth were tightly linked. Significant reductions in nucleotide pools that would be expected to affect growth were more evident in summer when pools were larger and growth was more rapid. But even where ATP and total adenine nucleotides were reduced, the ratio of ATP:ADP and the adenylate energy charge remained unchanged indicating an active adenylate kinase that had access to most of the adenine nucleotide pools, and possible catabolism of excess AMP.     

Effect of Abscisic Acid on the Transpiration of Zea mays

Intact plants of Zea mays L. were treated with foliar sprays of cis-trans-abscisic acid (ABA) at concentrations from 10⁻⁹ to 10⁻⁴M. Even the lowest concentration caused a reduction of the transpiration rate as measured between 1 and 33 h after spraying. With increasing ABA concentrations, there was a nearly linear relationship between the logarithm of the ABA concentration and the (decreasing) transpiration rate within that period. Subsequently a partial recovery of the transpiration rate set in, beginning progressively later as the ABA concentration was increased. After 5 1/2 days the transpiration rate of plants treated with 10⁻⁹ and 10⁻⁸M was nearly back to normal, whereas plants treated with 10⁻⁴M transpiration at only about 2/3 their normal rate. In experiments with detached maize leaves supplied with water or ABA solutions (10⁻⁸ to 10⁻⁵M) through their cut bases, the transpiration of control leaves decreased gradually to a low level in 24 h. ABA caused a marked and rapid reduction of the transpiration rate compared to that of the controls. After a few hours, the transpiration of the treated leaves decreased at a slower rate than that of the controls, thus approaching the control values. After 35 h, the transpiration of leaves treated with 10⁻⁵M ABA was nearly the same as in untreated leaves. Exchanging the ABA solution for distilled water after 24 h had little effect on the subsequent course of the transpiration rate.     

The Effect of High Concentrations of Heavy Water on Root Morphogenesis in Zea mays

The effect of high concentrations of heavy water on young rootsof Zea mays was investigated. Submersion for 24 hrs. in solutionsof 80–90 per cent. D₂O will temporarily stop growth ofthe primary root. During the treatment period the root-tip regionswill swell as the result of radial cell enlargement, primarilyin the cortical region. Upon removal from D₂O growth will resume,initially at a slower rate than controls but ultimately at similarmaximum rates. Lateral root production is inhibited in thatportion of the primary root which is the region of elongationat time of treatment. However, in the swollen region lateralroot formation is accentuated. The mitotic index drops sharplyupon introduction of the primary roots into 80 per cent. D₂O.Within 12 hrs. the index drops to zero and remains at this leveluntil the roots are removed from the heavy water. The mitoticindex then rises again, reaching control values within 24 hrs.It is suggested that the gentle, temporary inhibitory actionof D₂O makes this substance a useful tool in morphogenetic investigations.     

Metabolism of Proline, Glutamate, and Ornithine in Proline Mutant Root Tips of Zea mays (L.)

In excised pro_1-1 mutant and corresponding normal type roots of Zea mays L. the uptake and interconversion of [¹⁴C]proline, [¹⁴C]glutamic acid, [¹⁴C]glutamine, and [¹⁴C]ornithine and their utilization for protein synthesis was measured with the intention of finding an explanation for the proline requirement of the mutant. Uptake of these four amino acids, with the exception of proline, was the same in mutant and normal roots, but utilization differed. Higher than normal utilization rates for proline and glutamic acid were noted in mutant roots leading to increased CO₂ production, free amino acid interconversion, and protein synthesis. Proline was synthesized from either glutamic acid (or glutamine) or ornithine in both mutant and normal roots; it did not accumulate but rather was used for protein synthesis. Ornithine was not a good precursor for proline in either system, but was preferentially converted to arginine and glutamine, particularly in mutant roots. The pro_1-1 mutant was thus not deficient in its ability to make proline. Based on these findings, and on the fact that ornithine, arginine, glutamic acid and aspartic acid are elevated as free amino acids in mutant roots, it is suggested that in the pro_1-1 mutant proline catabolism prevails over proline synthesis.     

Root Cap Inhibitor Formation in Isolated Root Caps of Zea mays

Root caps were isolated and cultured aseptically on variousdefined media. Under appropriate culture conditions plus suitableillumination a substance able to produce a positive geotropicresponse (i.e. a downward bending of roots) was formed in isolatedroot caps. The presence of this substance, or root cap inhibitor,was assessed by substituting cultured caps in place of capsof dark-grown roots. Normally these roots if kept in continuousdarkness will not respond to gravity (i.e. no bending). Optimalroot cap inhibitor production occurs on a relatively simplemedium, lacking sucrose, but supplemented with 10^–9 MIAA. Protein synthesis is necessary for inhibitor productionand/or expression, whereas DNA synthesis is not.     

The Ultrastructure of the Regenerating Root Cap of Zea mays L

Removal of the cap from the primary root of Zea mays activatescell division in the quiescent centre. It is the descendentsof these cells that eventually regenerate a new cap—aprocess that is complete in about 4 days at 23 °C. The ultrastructureof the cells of the regenerating cap was examined at daily intervals.During the first day after decapping the dictyosomes in theexposed outer layer of cells change from a relatively quiescentstate to one where they are secreting a polysaccharide slimewhich accumulates between the plasmalemma and the outer cellwall. Amyloplasts grow in size and appear to divide, and theendoplasmic reticulum proliferates. Many different cytoplasmicfeatures that are normally characteristic of cells in distinctlocations within the undisturbed cap occur, at first, all togetherwithin the few cells that are the source of the new regeneratingtissue. Regeneration of a normal structure in the new cap isachieved by progressive changes in the structures of the cellorganelles, apparently in response to the position that thecells containing them occupy within the growing cellular ensembleat the root apex. Zea mays, regeneration, root cap, ultrastructure     

The Exchange of sodium Ions in the Root of Zea mays

The apparent cation-exchange capacity of maize roots has beenestimated in 100 mM NaCL and I mM NaCl by observing the effluxkinetics of isotopically labelled sodium from this tissue. Thisapproach indicated that the apparent capacity was 70 m.equiv/kgof tissue (fresh weight) and 2 m.equiv/kg of tissue (fresh weight)in 100 mM and I mM NaCl respectively; the pH of the medium was5.8 in each case.Similar sodium efflux experiments were performedon isolated stelar tissue from these roots and such experimentsgave an apparent exchange capacity of 86 m.equiv/kg of tissue(fresh weight) and 6 m.equiv/kg of tissue (fresh weight) in100 mM and I mM NaCl respectively, both at pH 5.8.The dependenceof the magnitude of the apparent exchange capacity upon theelectrolyte concentration of the external medium is discussed.     

Effects of Cycloheximide on Indoleacetic Acid-induced Ethylene Production in Pea Root Tips

Cycloheximide inhibited ethylene production in excised pea root tips treated with high levels of indoleacetic acid (100 μm and 10 μm). In contrast, cycloheximide did not inhibit ethylene production induced by a lower concentration (1 μm) of indoleacetic acid unless it was added 2 hours before the indoleacetic acid treatment. These observations suggest that indoleacetic acid has two effects on the enzyme system involved in ethylene synthesis. At low concentrations (1 μm) indoleacetic acid increases ethylene production without protein synthesis, whereas at the higher concentrations, the synthesis of new protein is associated with increased ethylene production.     

Root Growth Inhibitors from Root Cap and Root Meristem of Zea mays L.

A micro-assay based on the growth inhibition of root segmentsof the seminal roots of Zea mays has been used to investigatethe root-growth-inhibiting substances in root caps and meristemsrespectively of the roots of Zea mays. This micro-assay is sensitiveto 50 pg of IAA or less. Paper chromatography of the acid fractionof methanolic extracts shows the presence of one main inhibitorin root caps and a different main inhibitor in root meristems.Neither is IAA, whose presence in meristems is sometimes indicatedby small inhibitions (or stimulations) at the characteristicRf of IAA. A Commelina leaf-epidermis assay shows the presenceof one stomata-closing ABA-like substance in root caps and onein meristems, one corresponding in Rf to the main root-growthinhibitor from the root cap. The implications of these findingsfor the geotropic responses of roots is briefly discussed.     

Physiological Responses of Zea mays Seedlings to Interactions Between Cadmium and Salinity

The effects of 0, 2.5, 5.0, and 10.0 pmol/L Cd^2＋（Cd（NO3）2.4H2O） and 0, 10, 25, 50, and 100 mmol/L NaCl on growth, photosynthesis and the content of some ions in maize （Zea mays L.） were investigated in the present study. With Increasing concentrations of Cd^2＋ or NaCI alone in Hoagland nutrient solution, the chlorophylls and starch content decreased. Combination treatment with salinity and cadmium increased the negative effects observed following the two stresses alone. Plants exhibiting growth retardation in response to one mild stress factor （25-50 mmol/L NaCl） became more tolerant to the other stress factor （Cd）. The exposure of plants to cadmium caused a partial reversal of the effects of salinity. Root and shoot growth, ion accumulation and levels of photosynthetic pigments were improved at moderate concentrations of the two stress factors Imposed jointly.     

The Effect of the Conversion of Indolebutyric Acid into Indoleacetic Acid on Root Formation on Microcuttings of Malus

The uptake and metabolism of tritiated indolebutyric acid (IBA)and indoleacetic acid (IAA) were related to root regenerationon stem bases of apple (Malus cv "Jork") shootlets culturedin vitro. The major part of the auxins taken up from the mediumwas located in the bottom 1 mm of the stem basis, the locationwhere the roots emerge. In this part of the shoot about 4% ofthe accumulated IBA-³H remained in the free acid. Analysis onnormal phase TLC followed by reversed phase HPLC revealed thatabout 1% of the IBA-metabolites co-chromatographed with standardIAA. Incubation of shoots on medium with IAA led also to anIAA^int content of about 1% of the amount absorbed. IAA was notconverted into IBA. A medium concentration of 3.2 µM IAAor IBA induced maximum root formation of 9 and 13 roots pershoot, respectively. The IAA^int content in the stem base was0.5 µmol per kg FW after 5 days regardless of the auxinsource. Incubation on medium with IBA led to an IBA^int concentrationof 3.4 µmol per kg FW. IBA may exert its action partlyvia conversion into IAA. However, the fact that IBA inducedmore roots than IAA suggests that IBA itself is also active,or modulates the activity of IAA. The partition of absorbed auxin over active free auxin acidand individual conjugates was not directly related to root formation.At inductive and non-inductive auxin concentrations no shiftin the ratio of free auxin acids to total absorbed auxin wasobserved during root formation. (Received March 4, 1992; Accepted May 25, 1992)