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     

Biosynthesis of Indoleacetic Acid from Tryptophan-C in Cell-free Extracts of Pea Shoot Tips

A 2-step, 1-dimensional thin-layer chromatographic procedure for isolating indoleacetic acid (IAA) was developed and utilized in investigations of the biosynthesis of IAA from tryptophan-¹⁴C in cell-free extracts of pea (Pisum sativum L.) shoot tips. Identification of a ¹⁴C-product as IAA was by (a) co-chromatography of authentic IAA and ¹⁴C-product on thin-layer chromatography, and (b) gas-liquid and thin-layer chromatography of authentic and presumptive IAA methyl esters. Dialysis of enzyme extracts and addition of α-ketoglutaric acid and pyridoxal phosphate to reaction mixtures resulted in approximately 2- to 3-fold increases in net yields of IAA over yields in non-dialyzed reaction mixtures which did not contain additives essential to a transaminase reaction of tryptophan. Addition of thiamine pyrophosphate to reaction mixtures further enhanced net biosynthesis of IAA. It is concluded that the formation of indolepyruvic acid and its subsequent decarboxylation probably are sequential reactions in the major pathway of IAA biosynthesis from tryptophan in cell-free extracts of Pisum shoot tips. Comparison of maximum net IAA biosynthesis in extracts of shoot tips of etiolated and light-grown dwarf and tall pea seedlings revealed an order, on a unit protein N basis, of: light-grown tall > light-grown dwarf > etiolated tall etiolated dwarf. It is concluded that the different rates of stem elongation among etiolated and light-grown dwarf and tall pea seedlings are correlated, in general, with differences in net IAA biosynthesis and sensitivity of the tissues to IAA.     

Development of Vacuolar Volume in the Root Tips of Pea

Cell and vacuole areas were measured by light microscopy inlongitudinal and transverse sections cut at 0.4-mm intervalsalong the apical 7.2 mm of the primary root of pea. The vacuolararea (or volume) fraction — that is, vacuole area (orvolume) divided by cell area (or volume) — increased fromabout 15 % in cells 0.4 mm from the distal boundary of the apicalmeristem (the cap /root junction), to about 85% in cells situated6.8–7.2 mm from that boundary. At each distance, vacuoledevelopment tended to be greater in the cortex than in the stele.Vacuoles occupied about 22% of the tissue volume in the first1 mm length of root (measured from the cap/root junction), about31 % of the tissue volume in the first 2 mm, and about 45% whensummed over the apical 5-mm length of root. Phosphorus supplyor deprivation produced only minor and non-significant changesin vacuole development. The results have implications affectingprevious estimates of cytoplasmic and vacuolar phosphate concentrationsin pea root tips. Pisum sativum L., pea, root, vacuole, volume     

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.     

Ethylene Production in Pea and Cocklebur Seeds of Differing Vigour

Relationships between seed vigour and ethylene (C₂H₄) productionwere studied using C₂H₄-responsive fatty cocklebur seeds (Xanthiumpennsyhanicum Wallr.) and C₂H₄-insensitive starchy pea seeds(Pisum sativum L. cv. Alaska), which had been harvested in differentyears and subjected to different storage conditions. In bothspecies, the seeds with the highest vigour evolved the largestamounts of C₂H₄ during a period of water imbibition. The reductionof C₂H₄ production in cocklebur seeds occurred concomitantlywith the reduction in the growth potentials of both axial andcotyledonary tissues. Similarly, the activity of ACC-C₂H₄ conversionincreased with soaking, and was greater in seeds of high vigourcompared with those of low vigour. However, the change in ACCcontent in pea seeds differed from that in cocklebur seeds.That is, pea seeds with high vigour accumulated less ACC duringan imbibition period than those with low vigour. From theseresults it was suggested that the inferior C₂H₄ production bylow vigour pea seeds is mainly attributable to low ACC-C₂H₄conversion, whereas that by low vigour cocklebur seeds is dueto the shortage of ACC supply in addition to the reduced ACC-C₂H₄conversion. However, germination of deteriorated cocklebur seedswas not restored by exposure to ACC or C₂H₄, suggesting thatthe loss of seed vigour reduces the responsiveness of seedsto C₂H₄ as well as C₂H₄ production. Key words: Pea, cocklebur, seed vigour, ethylene production, 1-aminocyclopropane-1-carboxylic acid     

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.     

Ethylene Production by Root Nodules and Effect of Ethylene on Nodulation in Glycine max

Nodulated soybean roots produced more ethylene and contained more 1-aminocyclopropane-1-carboxylic acid than uninoculated roots. Nodules produced more ethylene and contained more 1-aminocyclopropane-1-carboxylic acid per gram of material than roots. Almost all of the ethylene produced by the nodules was produced by the plant fractions of the nodules. Ethylene, at physiological concentrations, did not inhibit nodulation in soybeans.     

Effect of Cycloheximide on Development of Mitochondria in Germinating Pea Cotyledons

Polyethylene glycol (PEG) mediated transfection of Lactobacillus casei ATCC 27092 protoplasts by phage PL-1 DNA was done. The protoplasts were obtained by treatment with purified PL-1 phage N-acetylmuramidase in the presence of citrate. Optimum conditions for transfection were 50% PEG 4,000, 15 µg protamine sulfate/ml, 0.15 m sucrose, and 10 m m MgSO₄ in MR medium (pH 6.0). The extent of transfection was proportional to the amounts of DNA added, and the greatest efficiency of transfection after a 10-min incubation was about 3.3 × 10⁵ PFU/µg DNA. The eclipse period of growth of progeny phages in the transfectants was 3 hr and the average burst size was 200.     

Effects of Dimethyl Sulfoxide and pH on Indoleacetic Acid-Induced Lateral Root Formation in the Radish Seedling Root

Segments (2.5 cm) cut from 3-day-old seedling roots of radish (Raphanus sativus L. `Scarlet Globe') were cultured in medium with or without indoleacetic acid (IAA). Lateral root primordia frequency, determined for the central centimeter of segments, was dependent on IAA concentration and on conditions affecting IAA uptake. Dimethyl sulfoxide treatment, or a relatively low medium pH, greatly enhanced the response to exogenous IAA. It was concluded that a permeation barrier exists between the external medium and the hormone responsive sites within the radish seedling root.     

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.     

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.     

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.     

Ethylene Effects in Pea Stem Tissue : EVIDENCE OF MICROTUBULE MEDIATION

The marked effects of ethylene on pea stem growth have been investigated. Low temperature and colchicine, both known microtubule depolymerization agents, reverse the effects of ethylene in straight growth tests. Low temperature (6 C) also profoundly reduces the effects of gas in terms of swelling, hook curvature, and horizontal nutation. Deuterium oxide, an agent capable of rigidifying microtubular structure, mimics the effects of ethylene. Electron microscopy shows that microtubule orientation is strikingly altered by ethylene. These findings indicate that some of the ethylene responses may be due to a stabilizing effect on microtubules in plant cells.     

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.