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.     

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.     

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.     

Inhibition of Acid-Enhanced Elongation of Zea mays Root Segments by Galactose

The effect of sugars and metabolic inhibitors on the elongation of Zea mays root segments was analyzed by a rhizometer which records the elongation of each of 32 root segments at the same time. Galactose suppressed the acid-enhanced rapid elongation after a lag period of 1.5 hours, but it did not inhibit the slow elongation at pH 7. Mannose was less inhibitory than galactose. Arabinose, xylose, glucose, sucrose, mannitol, and sorbitol caused no inhibition. When galactose was removed after a 1-hour treatment, the elongation was partially recovered. Cycloheximide and 2-deoxyglucose suppressed acid-enhanced elongation when these were applied at the same time as acid treatments, whereas cordycepin (3′-deoxyadenosine) inhibited elongation only if it was applied prior to acid treatment. Over the 9-hour period of elongation studied, the inhibition by galactose was comparable to that of cycloheximide. Since galactose has been reported to suppress the sugar metabolism necessary for the cell wall synthesis, the later phase of acid-enhanced elongation of root segments may at least partially depend on the synthesis or metabolism of cell wall components. The inhibition of root growth by galactose may be partially ascribed to a direct effect on the elongation process in roots, an effect that is enhanced by the acidification of the cell walls.     

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.     

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.     

Purine and Pyrimidine 5′-Nucleotide Catabolism in Tetrahymena pyriformis W1

SYNOPSIS Deamination at pH 7.5 of adenosine, deoxyadenosine, cytidine and deoxycytidine by cell-free preparations of Tetrahymena pyriformis W was observed both in the presence and absence of fluoride. Deamination of 5′-AMP, 5′-dAMP, 5′-CMP, and 5′-dCMP was found only in the absence of fluoride. Dephosphorylation of the above nucleotides by acid phosphatases occurred at pH 4.5; reduced activity was noted at pH 7.5. Fluoride effectively blocked acid phosphatase activity at both pH values. This correlation of phosphatase and deaminase activities suggests a catabolic pathway for 5′-AMP and 5′-CMP whereby dephosphorylation precedes deamination. Radiolabelled substrates were used to test this hypothesis. The experiments were designed so that conversion of as little at 1.0% of the radiolabelled substrate to the deaminated product could be detected. No 5′-IMP or 5′-UMP, the expected deamination products of 5′-AMP and 5′-CMP, respectively, was recovered after incubation of the radiolabelled substrates with cell-free enzyme preparations. Thus, it appears that Tetrahymena has no 5′-AMP or 5′-CMP deaminases and that these compounds are deaminated only after conversion to nucleosides. Acid phosphatase activity toward 5′-GMP, 5′-dGMP, 5′-TMP, 5′-UMP, and 5′-XMP was also found.     

The Movement and Physiological Effect of Indoleacetic Acid Following Point Applications to Root Tips of Zea mays

Pinpoint applications of labeled and non-labeled indoleacetic acid (IAA) on resin beads were made, without injury, to vertical roots of intact seedlings of Zea mays. Points of application were at the extreme tip of the root, 0.5, 2 and 5 mm from the root tip. The movement of label and bending of the roots was recorded. Radiolabel was found to move basipetally from the extreme tip and 0.5 mm applications to a similar extent, reaching 8 mm from the tip. The level of label in the growing zone after 4 h was 10% of that found in the extreme tip. Movement from 2 and 5 mm applications was equal in both directions. Higher amounts of non-labeled IAA caused bending towards the point of application if applied at 0.5 or 2 mm but not at 5 mm from the tip. It is proposed that any endogenous IAA in the root cap could move to the growing zone and cause a unilateral inhibition of growth, provided that it was in the same transport pool as the exogenously applied IAA.     

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.     

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.     

盐胁迫对玉米根尖质膜上受钙激活的蛋白激酶的影响

以02mal／L的NaCl对玉米根尖进行盐胁迫,导致质膜上一种受钙激活的蛋白激酶（该激酶表现为钙依赖蛋白激酶的特性）的活性迅速增加。盐胁迫至15min时激酶的活性达到最大,较对照高30％,以后逐渐降低,盐胁迫至切50min时激酶活性仍略高于对照。10％PEG6000处理玉米很尖也可诱导质膜上受钙激活的蛋白激酶活性增加。用外源ABA处理玉米根尖,未导致上述蛋白激酶活性的变化。放射自显影显示质膜上33kD和58kD两种蛋白可能是质膜受钙激活的蛋白激酶的内源底物。     

RNA Synthesis in the Root Apex of Zea mays

The rate of RNA synthesis in eight regions of the root apexof Zea mays has been determined. Cells of the quiescent centrehave a lower level of RNA synthesis than any other region studied.The level of RNA synthesis appears to be a function of the positionof the cell in the apex. All the newly synthesized RNA detectedin chromatin and cytoplasm can be accounted for by synthesisin the nucleolus and subsequent transport to these compartments.Newly synthesized RNA appears in the three compartments at differentrates depending on the location of the cell.     

Inhibition of RNA Synthesis and the Relationship between Nucleolar and Mitotic Cycles in Zea mays Root Meristems

The relationship between nucleolar and mitotic cycles has beendetermined after treatment of root apices of Zea mays with ethidiumbromide. In the meristematic regions of the stele the two cyclesare not much displaced in relation to each other except fora delay in the onset of the disorganization phase. A few nucleolipersist into metaphase and a few nuclei undergo an amitoticdivision. In the cap initials the drug greatly delays the onsetof disorganization of the nucleolus, which normally occurs beforeprophase in this region. It also delays the completion of reorganizationso that fully organized nucleoli are no longer available duringthe last half of telophase. In the quiescent centre the onsetof disorganization and the end of reorganization of the nucleoliare also delayed in relation to mitosis. There is no evidencefor a delay in the onset of reorganization in any region ofthe meristem. Some cells form multiple micronucleoli and this aberrant behaviouroccurs more often in the cap initials than elsewhere as doesamitotic division.     

Aluminum Exclusion Mechanism in Root Tips of Maize (Zea mays L.): Lysigeny of Aluminum Hyperaccumulator Cells

Abstract: Ultrastructural studies, together with X-ray microanalytical, immunocytochemical and cytochemical analysis performed in root tips of Al-resistant (C-525 M) and Al-sensitive (Adour 250, HS 7777 and BR 201 F) maize plants ( Zea mays L.), after 96 h exposure to 20 μM Al, showed qualitatively similar results in the four cultivars.
Al was identified in electron-opaque precipitates, which were insoluble even in EDTA chelate. They also contained an elevated proportion of P and also of K and Ca, some traces of Mn, Fe and Zn and sometimes of Mg. This elemental composition is similar to that described for phytin (Al-phytin), and the precipitates were localized in the two principal extraplasmatic compartments: cell walls and vacuoles. Al-phytin was detected in swollen areas of cell walls in membraneous concentric configurations, resembling myelin figures, probably rich in phosphatidyl inositol, which also intervene in the vacuolar internalization of Al-phytin and are similar to a peculiar form of endocytosis (not previously described). Abnormal apoplastic protuberances containing abundant electron-opaque Al-phytin deposits, agglutinated by callose (immunocytochemically identified), were shown in cortex cells with high mitotic activity (around 1 - 1.5 mm from cap root). Al-hyperaccumulator cells parallel to the root axis were correlated with longitudinal lysigenous intercellular spaces after cell death and dissolution (lysigeny). Indicators of activated lysigeny, as low levels of Al and callose (in agreement with other authors) and high levels of phosphoinositides, can mark Al-resistant genotypes, contrary to Al-sensitive genotypes, probably derived from a partially activated or even inactivated lysigeny.
The lysigeny of Al hyperaccumulator cells constitutes new ultrastructural evidence of an Al exclusion mechanism, supporting biochemical results reported by other investigators.     

不同盐处理对玉米幼苗根系质子分泌及细胞膜透性的影响

以玉米品种郑单958为实验材料,分别用100 mmol/L NaCl、100 mmol/L KCl和50 mmol/L Na2CO3处理其幼苗3 d,研究不同盐类对玉米根系质子分泌和细胞膜透性的影响.结果表明:不同盐处理都显著抑制玉米幼苗根系的生长,抑制程度依次为Na2CO3>KCl>NaCl;不同盐处理均使玉米幼苗根系Na 含量显著增加,NaCl和Na2CO3处理显著降低根系K 含量而导致其Na /K 升高,但KCl处理却显著提高根系K 含量使其Na /K 降低;不同盐处理均能显著增加细胞膜透性而降低根系质子分泌能力,影响程度依次为Na2CO3>KCl>NaCl.研究发现,相同阳离子浓度条件下,KCl处理对玉米根系质子分泌的抑制作用强于NaCl,碱性盐的抑制作用大于中性盐;盐胁迫可能通过改变玉米幼苗根系质膜的稳定性来影响质子分泌,从而抑制根系生长.     

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.     

Osmotic Stress Decreases the Activity of ATPase Associated with the Root Cap Plasmodesmata in Zea mays

With light and electron microscopy the substructural change and the ATPase activity of corn (Zea mays L. ) root cap cells after short-term osmotic stress were studied. Some spoke-like fine strands originating from the departed periplasm and stretching towards cell wall could be observed even after plasmolysis. By observing the precipitation of ATPase activity product (lead phosphate) at plasma membrane and plasmodesmata, it was found that the fine strands were plasma membrane-lined channels surrounding the cytoplasm and that they still firmly connected to the plasmodesmata during plasmolysis. Compared with the control (unstressed), a sharp decrease of ATPase activity in the plasmodesmata of the stressed cells was observed. Inhibition of energy metabolism in these limited locales would affect the physiological activity, maybe including the regulation of permeability and the change of size exclusion limit (SEL) of plasmodesmata.     

Root Morphology and Gene Expression Analysis in Response to Drought Stress in Maize (Zea mays)

Water-deficit stress tolerance is a complex trait, and water deficit results in various physiological and chemical changes in maize (Zea mays L.) and exacerbates pre-harvest aflatoxin contamination. The objective of this study was to characterize the variations in morphology, physiology, and gene expression in two contrasting inbred lines, Lo964 and Lo1016, in order to understand the differences in response to water-deficit stress. The results revealed that Lo964 was less sensitive to water-deficit stress, and had a strong lateral root system and a higher root/shoot ratio in comparison to Lo1016. In response to water-deficit stress by comparing stressed versus well-watered conditions, abscisic acid syntheses were increased in leaves, roots, and kernels of both Lo964 and Lo1016, but by different magnitudes. Indole-3-acetic acid (IAA) was undetectable in the leaves and roots of either genotype regardless of treatments, but increases of 58% and 8% in IAA concentration were observed in 20 DAP kernels, in response to water-deficit stress, respectively. The expression of the MIPS was up-regulated 7-fold in leaf tissues of Lo964 compared to Lo1016 at watered conditions, but decreased significantly to similar levels in both genotypes at water-deficit conditions. ZmPR10 and ZmFer1 expressions tended to up-regulate although ZmPR10 was expressed higher in root tissue while ZmFer1 was expressed higher in leaf tissue. Further study is needed to confirm if Lo964 has reduced aflatoxin contamination associated with the drought tolerance in the field in order to utilize the resistant trait in breeding.     

Suitability of the Osmotic Shock Procedure for the Analysis of Membrane Transport in Root Tips of Zea mays L.

Root tips of Zea mays L. previously subjected to osmotic shockwere studied as to uptake and efflux of glucose and uptake ofphosphate, as well as release of protein and other Lowry-positivesubstances. The osmotic shock procedure applied was similarto that frequently used in order to release periplasmic proteinsfrom bacteria. After treatment with hypertonic solutions of sorbitol (>0?3M) the uptake of phosphate and glucose is reduced and proteinis released into the medium. All osmotic treatments leadingto a significant reduction of either glucose or phosphate uptakeare accompanied by a strongly increased release of glucose andLowry-positive substances. Damage of plasma membrane barrierfunction can be directly observed by the uptake of Evans Blue(Gaff and Okong'o-Ogola, 1971) into the cells involving mainlyolder vacuolated rhizodermal cells. The occurrence and extent of the alterations of membrane semipermeabilityare strongly dependent on the duration of exposure to and theconcentration of the sorbitol solution used. The velocity ofthe osmotic transitions (from hypotonic to hypertonic and viceversa) is of minor importance. Sensibility of cells to osmotic stress increases with differentiationand vacuolation. Conditions for a selective reduction of phosphateuptake seem to occur when only meristem and near tip regionssubjected to a mild osmotic shock are used.