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.     

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.     

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.     

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)     

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.     

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.     

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.     

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.     

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).     

Distribution of Lateral Root Primordia in Root Tips of Musa acuminata Colla

The distribution of lateral root primordia in Musa acuminatashows discrete elements of pattern, a major element of whichis the rather regular spacing of laterals along protoxylem-basedranks. There is some co-ordination of positions of lateralsin different ranks. Laterals are apparently not initiated ina single acropetal sequence within the root tip as a whole althoughthey are initiated in acropetal sequence within each rank. Musa acuminata, banana, roots, lateral roots     

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.     

Morphactin and Adventitious Root Formation in Pea Cuttings

Stock plants of pea (Pisum sativum L. cv. Alaska) were grown at different controlled levels of irradiance (4, 16 or 38 W m^?2) for 11 days from sowing. Morphactin (CFM, methyl-2-chloro-9-hydroxy-fluorene-9-carboxylate) was applied to the apex of the stock plants 3 days before cuttings were excised. The cuttings were rooted at 16 W m^?2. High levels of morphactin (>5 × 10^?3 mg l^?1) inhibited root formation in the cuttings. Low concentrations of CFM (5 × 10^?5 mg l^?1) promoted the formation of adventitious roots in cuttings from plants grown at all three levels of irradiance, with the most pronounced effect in cuttings from 4 W m^?2. Measurements of ethylene evolution by CFM-treated plants 3 days after application, revealed a stimulatory effect on ethylene production by high CFM concentrations.     

Card Mounts for Handling Root Tips in the Paraffin Method

A method is described for utilizing card mounts to facilitate the handling of root tip samples and similar material in the paraffin method. The freshly collected roots are attached to temporary card mounts having dimensions of approximately 2 cm. × 2.5 cm. with DuPont Household Cement or a similar adhesive that hardens rapidly in the ordinary aqueous fixing fluids and is insoluble in the lower grades of alcohol. After fixation and dehydration to 75% alcohol, the roots are transferred to permanent card mounts on which they are carefully oriented for sectioning. Mucilage or glue which hardens rapidly in 85% alcohol and is insoluble in the ordinary dehydrating and infiltrating media is used in making the permanent card mounts. Detailed instructions are given for preparing and handling the card mounts, and a system of labeling the mounts is also suggested.     

Hemoglobin and Hypoxic Acclimation in Maize Root Tips

Class 1 hemoglobins (Hbs) have a wide distribution in the plant kingdom and have been demonstrated in root, seed, stem, and leaf tissues. They are present at low concentrations in aerobic tissue, but their synthesis is rapidly induced by hypoxic stress. The pattern of expression of the maize Hb gene in roots of young maize plants exposed to hypoxia has been examined. Root Hb gene expression increased rapidly to a maximum within first two hours of hypoxia, then declining to prehypoxia levels within 48-h hypoxic exposure. Limiting oxygen supply to the roots by total plant immersion and darkness did not alter the time course of hemoglobin expression. Hb gene expression was about 20-fold higher in the stele than in the cortex of control, aerobically grown roots. Stele Hb expression increased about fourfold under hypoxic conditions, whereas its expression in the cortex increased about 60-fold. In these samples, alcohol dehydrogenase (Adh) gene expression increased about four- and ten fold in the stele and cortex, respectively. The effect of the state of the Hb on anoxic survival of maize root tips was assessed by exposing root tips to a carbon monoxide atmosphere to maximize the proportion of hemoglobin in the carbonmonoxy form. Carbon monoxide had no significant effect on the survival or the ATP levels in anoxic maize roots, regardless of whether they had been acclimated by exposure to a hypoxic pretreatment. This would suggest that the presence of oxyhemoglobin is not essential for the survival of anoxic root tips.     

豌豆根胞质V1-ATPase的鉴定

利用免疫印迹、免疫电镜和ATP水解活性的测定对豌豆(Pisum sativum L.)根细胞胞质中V1-ATPase复合物的存在进行鉴定.用兔抗绿豆V-type H+-ATPase 的A、B亚基的抗体进行的immuno-blotting和胶体金电镜结果都表明,胞质中存在有A、B亚基.活性测定结果进一步表明胞质具有ATP水解活性.这些结果说明豌豆根胞质具有有活性的V1-ATPase复合物.这是首次直接证明植物中有胞质V1-ATPase的存在.