K+-Selective Inward-Rectifying Channels and Apoplastic pH in Barley Roots

Recent structure-function analysis of heterologously expressed K⁺-selective inward-rectifying channels (KIRCs) from plants has revealed that external protons can have opposite effects on different members of the same gene family. An important question is how the diverse response of KIRCs to apoplastic pH is reflected at the tissue level. Activation of KIRCs by acid external pH is well documented for guard cells, but no other tissue has yet been studied. In this paper we present, for the first time to our knowledge, in planta characterization of the effects of apoplastic pH on KIRCs in roots. Patch-clamp experiments on protoplasts derived from barley (Hordeum vulgare) roots showed that a decrease in external pH shifted the half-activation potential to more positive voltages and increased the limit conductance. The resulting enhancement of the KIRC current, together with the characteristic voltage dependence, strongly relates the KIRC of barley root cells to AKT1-type as opposed to AKT3-type channels. Measurements of cell wall pH in barley roots with fluorescent dye revealed a bulk apoplastic pH close to the pK values of KIRC activation and significant acidification of the apoplast after the addition of fusicoccin. These results indicate that channel-mediated K⁺ uptake may be linked to development, growth, and stress responses of root cells via the activity of H⁺-translocating systems.     

Apoplastic {alpha}-Amylase in Pea is Enhanced by Heat Stress

The apoplastic -amylase (EC 3.2.1.1 [EC] ) in pea (Pisum sativum L.cv. Laxton's Progress No. 9) seedlings was enhanced by a 4-to 6-fold with heat stress. Increased levels of the enzyme persistedfor 48 h at least after the stress and subsequent heat treatmentsenhanced the activity further. These results indicate that thefunction of apoplastic -amylase in vegetative tissues may berelated to plant stress. (Received October 14, 1996; Accepted February 20, 1997)     

Using Confocal Laser Scanning Microscopy to Measure Apoplastic pH Change in Roots of Lupinus angustifolius L. in Response to High pH

A confocal laser scanning microscopy method to measure apoplasticpH in situ of the tap roots of Lupinus angustifolius L. is described.Pseudo-ratiometric imaging of cell walls of lupin tap rootswas achieved by loading living roots with a pH-sensitive dye,Cl-NERF-dextran, and a pH-insensitive dye, Texas Red-dextran,as a reference. In 5-d-old tap roots which had previously beengrown at pH 5.4, the apoplastic pH in the elongating zone was5.2 ± 0.06. The apoplastic pH in roots grown at pH 7.5increased to 5.5. The method was validated by supplying differentforms of nitrogen in the nutrient solution. With     

Cadmium-Induced Inhibition of Apoplastic Ascorbate Oxidase in Barley Roots

The effect of excess cadmium (Cd: 0.0, 0.25, 0.5, 1.0 and 2.0 mM) on growth and ascorbate oxidase (AO) activity was investigated in barley (Hordeum vulgare L. cv. Jubilant) roots. The study employed a filter-paper technique to germinate and grow the germinating seeds following imbibition with respective Cd treatments for 4 h at 25 °C in darkness. Cd was required at 1.0 mM to affect 50% root growth inhibition 72 h after the treatment. This Cd-induced root growth inhibition was accompanied by a corresponding loss of plasma membrane integrity in root cells as evaluated by Evans blue uptake. Excess Cd (1.0 and 2.0 mM) significantly inhibited the AO activity in all the analysed fractions of barley roots such as extracellular, soluble, cell wall (CW)- and membrane-bound fractions. AO was localized in the apoplast, and its highest specific activity was detected in the CW II fraction obtained by extraction with 1.0 M NaCl from purified cell walls. The analysis of AO isozyme profile showed that besides the reduced activity of two anionic and two cationic isozymes, one cationic AO isozyme was activated during excess Cd treatment, which could be detected in cell wall fractions CW II, III and IV.     

Abscisic Acid Movement into the Apoplastic solution of Water-Stressed Cotton Leaves: Role of Apoplastic pH

Leaves of cotton (Gossypium hirsutum L.) were subjected to overpressures in a pressure chamber, and the exuded sap was collected and analyzed. The exudate contained low concentrations of solutes that were abundant in total leaf extracts, and photosynthetic rates and stomatal conductance were completely unaffected by a cycle of pressurization and rehydration. These criteria and others indicate that the experimental techniques inflicted no damage upon the leaf cells. The pH and abscisic acid (ABA) content of the apoplastic fluid both increased greatly with pressure-induced dehydration. Although ABA concentrations did not reach a steady state, the peak levels were above 1 micromolar, an order of magnitude greater than bulk ABA concentrations of the leaf blades. Treatment of leaves with fusicoccin decreased the K⁺ concentration, greatly reduced the pH rise, and completely eliminated the increase in ABA in the apoplast upon dehydration. When water-stressed leaves were pressurized, the pH of the exuded sap was increased by 0.2 units per 1 megapascal decrease in initial leaf water potential. Buffer capacity of the sap was least in the pH range of interest (6.5-7.5), allowing extremely small changes in H⁺ fluxes to create large changes in apoplastic pH. The data indicate that dehydration causes large changes in apoplastic pH, perhaps by effects on ATPases; the altered pH then enhances the release of ABA from mesophyll cells into the apoplastic fluid.     

Intervarietal Differences in the Amino Acid Composition of Pea Roots as Related to their Response to Salinity

The salt tolerance of three cultivars of Pisum sativum L. asdefined by root growth on saline substrate was in the orderDan > Alaska > Laxton Progress. The total content of freeamino acids decreased in Alaska, but not in Laxton Progress,with increasing salinity, while the content of the free basicamino acids increased with salinity. In both cultivars salinityinduced accumulation of free proline, but not enough to maintainthe osmotic adaptation of the cytoplasm. Proline accumulationcould not account for the greater tolerance of the Alaska cultivar.Both cultivars contain large amounts of glutamic acid and homoserine.The amino acid composition of protein of the Alaska cultivaris affected much more by salinity than is that of Laxton Progress.In Alaska salinity induces an increase in aspartic and glutamicacids, threonine, serine, leucine and isoleucine. The totalcontent of basic amino acids decreases with salinity. The proteinof Laxton Progress is very rich in leucine and its amount slightlydecreases with salinity. In the most tolerant cultivar, Dan,there is more proline in the protein than in the other two cultivarsand its content of leucine increases with salinity. The onlyfeatures which show some correlation with salinity toleranceare proline content (Dan > Alaska > Laxton Progress) andan increase in leucine content in the protein, with increasingsalinity. Pisum sativum L, pea, amino acid composition of roots, salinity     

Histopathology of Pea Roots Axenically Infected by Pratylenchus penetrans

The histological changes in pea roots axenically infected by Pratylenchus penetrans were studied and described. Roots of pea seedlings growing aseptically on the surface of nutrient agar slants were inoculated with axenized nematodes. Six hours after inoculation most of the nematodes introduced were probing the root epidermis, but none had completely entered though a few were observed with their anterior section already in the root. Most of the nematodes penetrated the roots after 12 hr inoculation. From 18 to 24 hr after inoculation the nematodes were mostly in the mid-cortex. Invaded regions of the cortex often showed orange discoloration. As incubation continued, the number of nematodes in these roots increased, and feeding and reproductive activities extended deeper into the cortex. These activities resulted in extensive breakdown of the cortex. No nematodes were observed within the stele of infected roots; however, the endodermis of infected roots stained dark-brown. Gravid female nematodes probed the root endodermis and some endodermal cells appeared to collapse after prolonged probing by the nematode. All stages in the life cycle of the nematode were observed in infected roots; the female to male ratio inside the root was about 5:1.     

Regulation of Apoplastic pH in Source Leaves of Vicia faba by Gibberellic Acid

Leaf discs of broad bean (Vicia faba L.), peeled on the spongy mesophyll side, rapidly altered the pH of the surrounding medium (apoplast). Using pH indicator paper appressed against the leaf, immediately after peeling, initial apoplastic pH was estimated to be 4.5. Changes in the apoplastic pH were measured with a microelectrode placed into a 100-microliter drop of an unbuffered solution (2 millimolar KCl, 0.5 millimolar CaCl₂, and 200 millimolar mannitol) on the peeled surface. Discs acidified the medium until the pH stabilized at about 5.0 (about 10 minutes). Acidification was inhibited by 50 micromolar sodium vanadate, an inhibitor of the plasmalemma H⁺-ATPase and attenuated by omitting the osmoticum or potassium ions from the medium. Fusicoccin (10 micromolar) greatly enhanced the rate of acidification. The presence of 0.1 to 1 micromolar gibberellic acid resulted in a slower rate of medium acidification. Gibberellic acid appeared to modulate the activity of the H⁺-translocating ATPase located at the plasma membrane of the mesophyll cells.     

Apoplastic pH of intact leaves of Vicia faba as influenced by light

The fluorochrome FITC-dextran was used to measure the effectof light on the apoplastic pH of intact Vicia faba leaves withthe ratio imaging technique. In darkadapted leaves the apoplasticpH varied depending on the leaf between 5.2 and 5.9. Red light(660 nm, 4–12 W m^–2) leads to multiphasic responses:in the first seconds an alkalinization ({small tilde}0.3 pHunits), and thereafter an acidification of the leaf apoplast({small tilde}0.4 pH units) were observed. Both effects couldbe inhibited by DCMU. While variation of CO₂ concentration revealedno effect on light-induced apoplastic pH changes, a decreasein O₂ concentration decreased the effect. On the basis of ourdata it is suggested that the influence of photosynthesis onplasmalemma H⁺ ATPase is responsible for the observed effects,rather than altered CO₂ uptake. Key words: Leaf apoplast, apoplastic pH, light, ratio imaging, pH-sensitive fluorescent dye, Vicia fab     

The Contribution of an Apoplastic Pathway to Sodium Uptake by Rice Roots in Saline Conditions

An apoplastic pathway, the so-called bypass-flow, across riceroots to the xylem has been investigated and approximately quantifiedusing the apoplastic tracer dye 8-hydroxy-l,3,6-pyrenetrisulphonicacid (PTS); former nomenclature 3-hydroxy-5,8,10-pyrenetrisulphonicacid. It was confirmed that PTS was confined to a compartmentno greater than the apparent free space in living rice roots.Experimental handling did not contribute to bypass-flow. Riceroots recovered rapidly from severe damage: following root pruning,sodium and calcium uptake returned to steady values in about6 h. Apoplastic flow declined after damage as a first-orderkinetic with a half time of 75 min. Analysis of the pattern of elution of PTS from preloaded roots(intact, excised and heat-killed), and from cellulose, was followedto compare PTS movement in the extracellular compartment withthat of water and small hydrated ions. Consideration is givento the factor by which the bypass-flow estimated with the dyewould need to be corrected to reflect the proportion of thetranspiration stream flowing in the apoplastic pathway. Thedata suggest that this factor would be at least 10 for transpiringrice plants. There was large individual variation in the transport both ofsodium and of the apoplastic tracer PTS to the shoot. Plantswith high sodium transport also had high PTS transport and itis concluded that some proportion of the sodium reaching thexylem in rice does so by a pathway which is also available toPTS, presumably direct apoplastic contact across the endodermis.A median value for the bypass-flow of water (corrected fromPTS) would be 0.5 to 1.0 percent of the transpirational volumeflow, but individuals with the highest sodium transport wouldhave bypass-flow values of several percent. Evidence is discussedwhich suggests that apoplastic transport may increase in stressconditions, and it is argued that bypass-flow is a major contributionto sodium uptake in rice in saline conditions. Key words: Oryza saliva, salinity, roots, radial ion transport, apoplast, bypass-flow     

ABA in Roots and Leaves of Flooded Pea Plants

Zhang, J. and Davies, W. J. 1987. ABA in roots and leaves offlooded pea plants.—J. exp. Bot. 38: 649–659. Roots of potted pea (Pisum sativum L. cv. Feltham First) seedlingswere flooded with tap water. Within a few hours of the startof the flooding treatment the content of free ABA in roots increasedcompared to contents of roots of unflooded control plants butthis increase was not statistically significant until the beginningof the second day after flooding. Approximately 36 h after firstflooding significant increases in the free-ABA content of leaveswere detected. This was 14 h after significant increases inthe amount of ABA in the roots of the same plants. There wasmarked diurnal variation in free-ABA content of leaves and rootsof plants that had been flooded for several days, with maximumcontents recorded 3 h or more after the beginning of the lightperiod. Very rapidly after the lights were switched oft ABAcontents declined. On day 3 of the flooding treatment, therewas more than a 5-fold decrease in the free-ABA content of leaveswithin a few hours of the beginning of the dark period. Radio-immunoassaysuggested that a very large proportion of the total ABA in theplant was in a bound form. This form of ABA increased substantiallyas the flooding period progressed. The importance of variation in ABA content for the control ofwater relations and gas exchange of flooded plants is discussed. Key words: Flooding, Pisum sativum, ABA, water relations     

Chlorophyll Formation in Excised Roots of Cucumber and Pea

The effects of red and blue light on the formation of chlorophyll in excised roots of cucumber and pea was investigated and compared with previously reported experiments on wheat roots. All three kinds of roots are similar in that in continuous red light no or very little chlorophyll is formed. and in blue light a considerable amount of chlorophyll is produced. Roots of cucumber and pea differ from those of wheat, in that red light is ineffective (or nearly so) even when combined with blue.     

Azelaic Acid-Induced Enzymes of Phenolic Defense in Pea Roots

The treatment of pea roots with azelaic acid for 72 h led to a change in the content of 28 proteins: the content of 20 proteins decreased, and the content of 8 proteins (including the phenylpropanoid metabolism enzymes, which are involved in the synthesis of phytoalexins) increased.     

Evaluation of Commercial Fluorescein Isothiocyanates Used in Fluorescent Antibody Studies

Commercial preparations of fluorescein isothiocyanate (FITC) for immunofluorescence applications were obtained from 12 sources and examined for purity by quantitative infrared spectrophotometry and by labeling efficiency for bovine serum albumin (BSA). Quantitative photometric measurements were made of nonspecific staining (NSS) produced by conjugates prepared from the dyes. The purity of FITC from different sources was highly variable. The risk of NSS appears to increase as the purity of the dye decreases. In immunofluorescence applications it is desirable to use the purest FITC available in order to obtain conjugates with minimum NSS. It is recommended that 70% FITC, as determined by BSA labeling efficiency, be accepted as the minimum purity for immunofluorescence applications.     

The Exodermal Casparian Band of Onion Roots Blocks the Apoplastic Movement of Sulphate Ions

Peterson, C. A. 1987. The exodermal Casparian band of onionroots blocks the apoplastic movement of sulphate ions.—J.exp. Bot. 38: 2068–2081. It has previously been established that the exodermal Casparianbands in the roots of onion and many other angiosperm speciesare impermeable to fluorescent, apoplastic dyes. The permeabilityof the onion root exodermal Casparian band to ions was testedby measuring sulphate free space in the walls using compartmentalanalysis by elution. Results of this analysis revealed fourcompartments for sulphate. The first was a surface film, between15 and 35 µm thick, which had a half-time of elution tooshort to be measured (i.e. less than 15 s). The second was awall compartment with an average half-time of elution of 1·0min. The third and fourth compartments were the cytoplasm andvacuole which had half-times of elution of 12·4 min and4·9 d, respectively. Experiments in which the temperatureand concentration of sulphate in the treatment solution werevaried indicated that the compartment identified as the wallwas a free space. Segments were excised from roots in regionswith a mature exodermal Casparian band. The sulphate free spacein the walls of ‘whole’ segments (i.e. segmentswith their cut ends sealed) was compared with that of ‘bisected’segments (i.e. segments which had been bisected and their stelesremoved). The free space in the walls of the whole segmentswas consistently smaller than the comparable free space in thebisected segments; the ratio whole/bisected ranged from 0·16to 0·26. This ratio compared favourably with predictedvalues obtained by taking the ratio of the wall volumes, i.e.walls external to the exodermal Casparian band/walls internaland external to the exodermal Casparian band. The ratios predictedfrom wall measurements varied from 0·25 to 0·27.Furthermore, the volume of the sulphate free space in the wholesegments was less than the volume of the walls external to theCasparian band. The results of the present study show that theexodermal Casparian band of onion is impermeable to sulphateions, which have a hydrated ionic radius of 0·378 nm. Key words: Allium cepa, exodermis, sulphate     

Apoplastic pH and Ammonium Concentration in Leaves of Brassica napus L

A vacuum infiltration technique was developed that enabled the extraction of apoplastic solution with very little cytoplasmic contamination as evident from a malate dehydrogenase activity of less than 1% in the apoplastic solution relative to that in bulk leaf extracts. The volume of apoplastic water, a prerequisite for determination of the concentration of apoplastic solutes, was determined by vacuum infiltration of indigo carmine with subsequent analysis of the dilution of the dye in apoplastic extracts. Indigo carmine was neither transported across the cell membrane nor significantly adsorbed to the cell walls, ensuring reproducible (SE < 2%) and precise determination of apoplastic water. Analysis of leaves from four different positions on senescing Brassica napus plants showed a similar apoplastic pH of 5.8, while apoplastic NH4+ increased from 1.1 mM in lower leaves to 1.3 mM in upper leaves. Inhibition of glutamine synthetase in young B. napus plants resulted in increasing apoplastic pH from 6.0 to 6.8 and increasing apoplastic NH4+ concentration from 1.0 to 25.6 mM, followed by a marked increase in NH3 emission. Calculating NH3 compensation points for B. napus plants on the basis of measured apoplastic H+ and NH4+ concentrations gave values ranging from 4.3 to 5.9 nmol NH3 mol-1 air, consistent with an estimate of 5.3 [plus or minus] 3.6 nmol NH3 mol-1 air obtained by NH3 exchange experiments in growth chambers. A strong linear relationship was found between calculated NH3 compensation points and measured NH3 emission rates in glutamine synthetase-inhibited plants.