Observation of Cytoplasmic and Vacuolar Malate in Maize Root Tips by C-NMR Spectroscopy

The accumulation of malate by maize (Zea mays L.) root tips perfused with KH¹³CO₃ was followed by ¹³C nuclear magnetic resonance spectroscopy. In vivo nuclear magnetic resonance spectra contained distinct signals from two pools of malate in maize root tips, one at a pH ~5.3 (assigned to the vacuole) and one at a pH > 6.5 (assigned to the cytoplasm). The ratio of cytoplasmic to vacuolar malate was lower in 12 millimeter long root tips than in 2 millimeter root tips. The relatively broad width of the signals from C1- and C4-labeled vacuolar malate indicated heterogeneity in vacuolar pH. During the 3 hour KH¹³CO₃ treatment, ¹³C-malate accumulated first primarily in the cytoplasm, increasing to a fairly constant level of ~6 millimolar by 1 hour. After a lag, vacuolar malate increased throughout the experiment.     

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.     

DNA Replication-Dependent Histone H2A mRNA Expression in Pea Root Tips

Histone H2A mRNA is selectively expressed in scattered subpopulations of cells in the pea (Pisum sativum) root apical meristem. To study whether this specific expression was associated with the cell cycle, a double-labeling technique was used to identify cells replicating DNA during S phase and those expressing H2A mRNA. Cells in S phase were detected by [3H]thymidine incorporation and autoradiography, whereas cells containing H2A mRNA were identified by in situ hybridization using digoxigenin-labeled probes. Approximately 92% of the [3H]thymidine-labeled S-phase cells expressed H2A mRNA and 85% of cells that expressed H2A mRNA were in S phase. In root tissue located basal to the promeristem, synchronous co-located expression was observed in scattered packets of proliferating cells. Furthermore, neither H2A mRNA nor S-phase cells could be detected within the quiescent center or mature root cap. When DNA synthesis was inhibited with hydroxyurea, a commensurate and specific decrease in steady-state levels of H2A mRNA was found. We conclude that cell-specific expression of pea histone H2A mRNA is replication dependent and that H2A mRNA is transiently accumulated during a period of the cell cycle that mostly overlaps the S phase. We propose that the overlap between H2A expression and S phase could occur if H2A mRNA accumulation began in late G1 and abated in late S.     

Regulation of Cytoplasmic and Vacuolar pH in Maize Root Tips under Different Experimental Conditions

³¹P-Nuclear magnetic resonance spectra of perfused maize (Zea mays L., hybrid WW x Br 38) root tips, obtained at 10-minute intervals over 12 hours or longer, indicate that no cytoplasmic or vacuolar pH changes occur in these cells in the presence of 25 millimolar K₂SO₄, which induces extrusion of 4 to 5 microequivalents H⁺ per gram per hour. In contrast, hypoxia causes cytoplasmic acidification (0.3-0.6 pH unit) without a detectable change in vacuolar pH. The cytoplasm quickly returns to its original pH on reoxygenation. Dilute NH₄OH increases the vacuolar pH more than it does the cytoplasmic pH; after NH₄OH is removed, the vacuole recovers its original pH more slowly than does the cytoplasm. The results indicate that regulation of cytoplasmic pH and that of vacuolar pH in plant cells are separate processes.     

Effect of Chloride and Sulphate Types of Salinity on the Nicotinamide-adenine-dinucleotides in Pea Root Tips

It was shown that in pea root tips grown in media salinatedwith NaCl the content of NAD + NADH decreased while the contentof NADPH+NADP increased with increasing salinity. In pea roottips, grown in sulphate salinated media, only the decrease inthe content of NAD+NADH was noted. The content of NADPH+NADPin such root tips remained more or less constant. The implicationof these results and of previous results for the explanationof the nature of salinity damage was discussed.     

Analysis of Cytosine Methylation Pattern in Response to Water Deficit in Pea Root Tips

Abstract: The correlation between environmental stress and DNA methylation has been studied by following the methylation status of cytosine residues in the DNA of pea root tips exposed to water deficit. DNA methylation was evaluated by two complementary approaches: (i) immunolabelling by means of a monoclonal antibody against 5-methylcytosine; (ii) MSAP (Methylation-Sensitive Amplified Polymorphism) to verify if methylation and de-methylation in response to water deficit may be related to specific DNA sequences. Immunolabelling showed that water stress induces cytosine hypermethylation in the pea genome. Regarding the CCGG target sequence, an increase in methylation specifically in the second cytosine (about 40 % of total site investigated) was revealed by MSAP analyses. In addition, MSAP band profile detected in three independent repetitions was highly reproducible suggesting that, at least for the CCGG target sequence, methylation was addressed to specific DNA sequences.     

Differential Expression of Proteins and mRNAs from Border Cells and Root Tips of Pea

Many plants release large numbers of metabolically active root border cells into the rhizosphere. We have proposed that border cells, cells produced by the root cap meristem that separate from the rest of the root upon reaching the periphery of the cap, are a singularly differentiated part of the root system that modulates the environment of the plant root by producing specific substances to be released into the rhizosphere. Proteins synthesized in border cells exhibit profiles that are very distinct from those of the root tip (root cap, root meristem, and adjacent cells). In vivo-labeling experiments demonstrate that 13% of the proteins that are abundant in preparations from border cells are undetectable in root tip preparations. Twenty-five percent of the proteins synthesized by border cells in a 1-h period are rapidly excreted into the incubation medium. Quantitative variation in levels of specific marker proteins, including glutamine synthetase, heat-shock protein 70, and isoflavone reductase, also occurs between border cells and cells in the root tip. mRNA differential-display assays demonstrate that these large qualitative and quantitative differences in protein expression are correlated with similarly distinct patterns of gene expression. These observations are consistent with the hypothesis that a major switch in gene expression accompanies differentiation into root border cells, as expected for cells with specialized functions in plant development.     

Water Deficit in Pea Root Tips: Effects on the Cell Cycle and on the Production of Dehydrin-like Proteins

Dehydrin-like proteins have been detected in nuclei and cytoplasmof meristematic root tip cells from pea seedlings subjectedto slow dehydration at 90% relative humidity for 48 h or more.Evidence was gained from Western blotting and immunocytochemicalexperiments using an antibody raised against the conserved domainof dehydrin proteins. Flow cytometer analysis has shown thatcycling cells of root tip meristems from dehydrated seedlingsare mostly arrested in G2 phase. Other stress treatments thoughtto involve water depletion (osmotic stress, cold treatment)or to modulate cell response to water deficit (abscisic acid)gave less clear-cut results with all treatments lowering theproportion of cells entering the S phase, but without a definiteand persistent arrest in any preferential phase of the cycle.Possible interrelationships between G2 arrest and dehydrin productionare discussed. Cell cycle; dehydrins; flow cytometry; nuclei; pea; Pisum sativum L.; water stress     

Physiological Role of Leghaemoglobin Heterogeneity in Pea Root Nodule Development

Disk-gel-electrophoresis of the leghaemoglobin isolated frompea root nodules revealed two major (Lb I and Lb IV) and threeminor components. The ratio of the two major components (LbI/Lb IV) decreased with increasing age. This ratio was higherin the distal than in proximal region when nodules were cutinto distal and proximal sections. In young nodules, the incorporationof radioactive leucine into Lb I was much higher than into LbIV. In older nodules, the radioactivities were much higher inLb IV than in Lb I. These data suggest that the change in thisratio is due mainly to differences in the rates of biosynthesis. Two major components (Lb I and Lb IV) which were isolated separatelyhad different O₂-binding affinities. The O₂-binding affinityof the component synthesized mainly in older nodules (Lb IV)was higher than that of the component synthesized mainly inyoung nodules (Lb I). These results indicate that changes inthe relative contents of leghaemoglobin during nodule developmentcontribute to more effective nitrogen fixation which is mediatedby changes in the capacities of oxygen transport. (Received July 7, 1981; Accepted November 2, 1981)     

The Chemical and Mechanical State of the Cell Wall of Pea Root Tips: I. PRELIMINARY OBSERVATIONS

The separation of pea root-tip cells after treatment with acidwas studied by the use of a mechanical device that squashedthe tissues under a constant load. The increase in area of thetissue as a result of the squashing was used as a quantitativemeasure of the cohesive forces that held the cells together. Interruption of the tissue respiration prior to the testing,by incubation under nitrogen for a period of 15 minutes or longer,led to a substantial increase in the acid-resistant mechanicalstrength, as was also the case if respiratory inhibitors wereadded to root tips during incubation under air. It was alsofound that this response varies in serial segments of the root,being greatest in the actively growing regions. The nature of this change in the state of the tissue is discussed,and the conclusion is drawn that in the natural state of thetissues a mechanism linked to the respiratory processes mustbe responsible for maintaining the tissues in a state susceptibleto treatment with acid.     

Root Formation in Pea Cuttings

The importance of the active shoot meristems for root formation in cuttings has been investigated through disbudding, decapitation or both disbudding and decapitation of pea cuttings at different time intervals after the removal of the cutting. Decapitation and disbudding within the first 4 days after cutting drastically reduce both the number of rooted cuttings and the number of roots per cutting. Treatment 5 to 6 days after cutting has little or no effect on the root formation or the number of roots per cutting. Redaction in rooting is explained by the removal of the production center(s) for the growth promoters which are necessary for root formation. It is deduced from the results obtained that the initiation phase in pea cuttings is about four days. The author is much indebted to the Department of Plant Physiology of the Royal Veterinary and Agricultural University, Copenhagen, for permission to use its growth chambers during the present investigations.     

Root Formation in Pea Cuttings

Cuttings were either decapitated or both decapitated and disbudded at different time intervals. Auxin, at different concentrations, was applied to the cuttings in lanoline. Auxin applied to decapitated and disbudded cuttings promoted root formation in the early stage of the initiation phase. Auxin treated cuttings, which were only decapitated, did not show an increase in number of roots per cutting. However, an increase in the root mass was found in the early stage of the initiation phase. The results seem to indicate that auxin is active only in the first part of the initiation phase. It is acting alone, not together with other substances synthesized in the shoot meristem.     

Root Formation in Pea Cuttings

Auxin was applied to the upper part of the cuttings, which were both decapitated and disbudded on the same day. The applied auxin was removed by redecapitating the cuttings at different time intervals. In a second experiment, auxin was applied either to the upper or lower part of the decapitated and disbudded cuttings at different time intervals. In cuttings, which were redecapitated after 1 and 2 days, the root formation was reduced considerably. The redecapitation after 3 days had no adverse effect on the root formation. Cuttings treated with auxin at different time intervals showed a weaker root promotion on days 0 and 1 than on the subsequent days. The results emphasize the fact that auxin is active only during the first part of the root initiation phase. A continuous flow of auxin for a period of the first 3 days during the root initiation is of overriding importance. There appears to be at least two different stages of the root initiation phase, (ia) auxin active stage, and (ib) auxin inactive stage. The results also seem to indicate that some other factors, in addition to auxin, are active during the first stage of the root initiation phase.     

Phosphorus Nutrition and the Intracellular Distribution of Inorganic Phosphate in Pea Root Tips: A Quantitative Study using31P-NMR

Pea plants (Pisum sativum L.) were supplied with external phosphatefor differing periods of time, so that their phosphorus statusvaried, and the intracellular distribution of inorganic phosphate(P₁) in the roots was examined by ³¹P nuclear magnetic resonance.Over the range of phosphorus nutrition investigated, the quantityof vacuolar P₁ per unit fresh weight of root tip changed considerably,whereas the quantity of cytoplasmic P₁ per unit fresh weightof root tip did not alter. The relative volumes of the cytoplasmand the vacuole in pea root tips seemed to be little affectedby differences in phosphorus nutrition, and this implied thatthe concentration of P₁ in the cytoplasm was kept almost constant,at a level estimated to be 18 mM. The rate of absorption of ³²P-labelled phosphate was negativelycorrelated with the vacuolar P₁ concentration, but there wasno clear correlation with the concentration of P₁ in the cytoplasm. Key words: Compartmentation, Cytoplasm, Vacuole, Concentration, Absorption     

Distribution of Lateral Root Primordia in Root Tips of Musa

The distribution of lateral root primordia in the root tipsof four Musa landraces (Grande Naine, Pisang Berlin, Ngok Egomeand Yangambi Km5) grown in the field has been investigated toevaluate the range of genetic variation of lateral root initiation.In banana (Musa sp.), lateral roots are initiated in the roottip, 0.6–4 mm behind the root/cap junction and arise inseveral protoxylem-based longitudinal rows or ‘ranks’.Significant differences were observed among landraces for theposition of the most distal primordium, however the longitudinalspacing between successive primordia along the ranks was similarfor all landraces. All ranks were involved in lateral root initiation.The number of ranks also showed significant variations amonglandraces and was proportional to the stelar diameter. Hencethe density of lateral roots (roots cm^-1) was affected by stelardiameter variations. Finally, root elongation in the root tipwas landrace-specific and not necessarily exponential, unlikesuggested in previous studies. It is concluded that lateralroot initiation in Musa is not involved in the genetic variationsof root architecture in the field. A dissection of root architectureinto components which may account for these variations is proposedin relation to the improvement of root system architecture.Copyright 1999 Annals of Botany Company Lateral root initiation, root architecture, Musa, banana.     

Immunocytochemical Localization of the Vacuolar H-ATPase in Maize Root Tip Cells

The vacuolar H⁺-ATPase of maize (Zea mays L.) root tip cells has been localized at the EM level using rabbit polyclonal antibodies to the 69 kilodalton subunit and protein A-colloidal gold. Intracellular gold particles were detected mainly on the tonoplast and Golgi membranes. Only about 27% of the vacuoles were labeled above background. The absence of gold particles on the majority of vacuoles suggests either that the tonoplast H⁺-ATPase is degraded during tissue preparation or that the small vacuoles of root tip cells are specialized with respect to H⁺-ATP ase activity. The pattern of gold particles on the labeled vacuoles ranged from uniform to patchy. Virtually all of the Golgi bodies were labeled by the antibody, but the particle densities were too low to determine whether the H⁺-ATPase was associated with specific regions, such as the trans-face. Cell wall-labeling was also observed which could be partially prevented by the inclusion of gelatin as a blocking agent. The immunocytochemical results confirm previous biochemical studies with isolated membrane fractions (A Chanson, L Taiz 1985 Plant Physiol 78: 232-240).     

Root Formation in Pea Cuttings III.

Cuttings were either decapitated or both decapitated and disbudded at different time intervals. Cytokinin, at different concentrations, was applied to the cuttings in lanoline. Higher concentrations of cytokinin inhibited root initiation during the early stage. However, the inhibitory effect of cytokinin disappeared during the later stage of root initiation. Lower concentrations of cytokinin promoted the root initiation during the early stage. This effect was observed on cuttings which were only decapitated. These results seem to indicate that the influence of cytokinin changes with the stage of development. There seems to be an interaction between cytokinin and one or more other growth factors. A possible reason for this may be that cytokinin, in higher concentrations, produces inhibitory effects during the early part of root initiation by blocking the activity of auxin. The loss of the inhibitory effect of cytokinin during the later part of the initiation phase suggests that, at this stage, developing root primordia are capable of controlling the level of active cytokinin and thus do not react to the exogenous application of cytokinin.     

The Chemical and Mechanical State of the Cell Wall of Pea Root Tips: II. THE EFFECTS OF SOME METABOLIC CHANGES

The area covered by a tissue-squash prepared by a standard procedurefollowing an acid treatment was taken as a quantitative inversemeasure of the intercellular cohesion. The effect on this acid-resistantcohesion of prior incubations in buffered (5x10^-5M. to 5x10^-13M.)ß-indolylacetic acid, sucrose (2.3 per cent w/v),and thioglycollate (10^-1 M. to 10^-4 M.) was studies, and alsothe effect of performing these incubations under nitrogen. Theauxin and sucrose treatments resulated in a weakening of theacid-resistant cohesion, and the thioglycollate incubation impededthe increase in acid-resistant cohesion induced by anaerobiosis. The results are discussed in terms of chemical changes thatmust occur in the structure of the middle lamella.