P-NMR Spectroscopy of Roots of Intact Corn Seedlings

The application of ³¹P nuclear magnetic resonance spectroscopy to the study of metabolism in roots of intact corn seedlings is described. ³¹P-NMR spectra of developmentally distinct parts of primary roots of whole seedlings are presented. The spectra are of quality comparable to those of excised pieces of plant tissue.     

Water Transport in Isolated Maize Roots

A simple model has been devised which predicts the concentration,C^x_s, of salt (e.g. KCl) in the exudate from isolated roots asa function of the salt concentration, C⁰_s, in the medium. Thechief assumption, made in deriving the relationship betweenC^x_s and C⁰₅, is that the exudation of water, J, from the rootsconsists of two components (one being osmotic, Ø, inorigin and the other, Ø⁰, flowing in the absence of anosmotic gradient). The exudation of salt, J_s, calculated asJ C^x_s, was found to be dependent on C⁰_s. Our investigationson maize roots were concerned with estimations of L_p and Ø^vand determinations of C^x_s as a function of C⁰_s. Satisfactoryagreement between prediction and experiment was found in thesepreliminary studies. It is considered that water movement inisolated roots can be explained by a simple osmotic model withthe additional possibility that a relatively small non-osmoticwater flow occurs.     

Determination of Unidirectional Sodium Fluxes in Roots of Intact Sunflower Seedlings

The method of compartmental analysis was applied to study sodiumfluxes in roots of intact seedlings of Helianthus annuus L.By measuring sodium uptake and transport to the shoots of theseedlings in parallel experiments, transport of tracer sodiumto shoots and net accumulation of Na⁺ in the roots during theflux measurements was accounted for. The steady-state sodiumfluxes in the intact sunflower roots were similar in size tothose in excised roots but in general they were somewhat higher.This indicates more metabolic activity in the intact tissues.Using whole plants it is possible to study the response of ionfluxes in roots to ecophysiological stimuli received by theshoots, and in the present experiments the effect of continuouslight versus long-day growth conditions was investigated. Potassium,when continually present, depressed all fluxes and the cytoplasmiccontent of sodium but tended to increase the vacuolar sodiumcontent, in particular when this was related to the cytoplasmiccontent. When added to sodium-loaded roots, potassium stimulatedthe plasmalemma sodium efflux but slightly, suggesting a lowefficiency of K⁺-Na⁺ exchange across the plasmalemma in intactas well as excised sunflower roots. Subsequently, however, potassiuminduced a transient decrease in the ²²Na efflux that was followedby oscillations in tracer efflux. These changes were attributedto potassium-induced transfer of sodium to vacuoles. Moreover,the oscillations seem to indicate the operation of negativefeedback control of sodium fluxes.     

Acidification of Deionized Water by Roots of Intact Rice Seedlings

This investigation was designed to examine whether or not deionizedwater could be acidified by roots of intact rice seedlings.Roots of intact rice seedlings caused significant acidificationof the deionized water in which they were immersed and thisacidification could be repeated after replacement of acidifiedwater with fresh deionized water. The addition of K⁺, Na⁺, andMg²⁺ to the deionized water significantly increased the rateand extent of acidification. However, no such increase was foundwhen Ca²⁺ was present in the water. The inhibition of acidificationby vanadate and its promotion by fusicoccin indicated that theacidification of water by roots of intact rice seedlings originatedfrom an ATP-driven proton pump located in the plasmalemma. Ferricyanide was effectively reduced by the roots of intactrice seedlings. This reduction was associated with the acidificationof the bathing solution. 8-Hydroxyquinoline and p-nitrophenyl-acetateinhibited both the reduction of ferricyanide and ferricyanide-inducedacidification. Vanadate, although it slightly inhibited thereduction of ferricyanide, did not inhibit the ferricyanide-stimulateddecrease in pH. It seems that the involvement of redox activityassociated with the plasmalemma in the acidification of deionizedwater cannot be excluded. (Received August 30, 1989; Accepted April 5, 1990)     

NaCl胁迫对茄子嫁接苗根系多胺代谢的影响

采用高压液相色谱法对80 mmol.L-1NaCl胁迫下营养液栽培茄子嫁接苗和自根苗根系多胺代谢的差异进行了研究。结果表明,胁迫2 d时,嫁接苗根系游离态亚精胺和结合态多胺含量显著高于自根苗,游离态腐胺和束缚态多胺显著低于自根苗,游离态精胺含量两者无显著差异。胁迫10 d时,自根苗3种形态的多胺含量均下降,显著低于对照植株,而嫁接苗根系游离态精胺、结合态腐胺和束缚态多胺含量显著高于对照植株。研究结果表明,NaCl胁迫显著提高了茄子嫁接苗和自根苗根系多胺氧化酶的活性,且自根苗增加幅度显著高于嫁接苗;嫁接苗胁迫初期根系结合态多胺的迅速积累及胁迫后期保持较高的精胺含量有利于其耐盐性的提高。     

Axonal Transport of Polyamines in Intact and Regenerating Axons of the Rat Sciatic Nerve

The axonal transport of putrescine or its polyamine derivatives spermidine or spermine is a subject of some debate. We investigated this question by injecting [3H]putrescine into the lumbar spinal cord of the rat and measuring the accumulation of radioactivity central to ligatures placed on intact and regenerating sciatic nerves. In normal nerves, approximately twice as much radioactivity built up proximal to these ligatures 2 or 3 days after injection than at more distal ligatures used to control for accumulation of radioactivity which might be due to tissue damage alone. In regenerating nerves the amount of radioactivity accumulating at the ligature was approximately five times that at the distal ligature and two to three times greater than in intact nerves. The identity of the radioactivity in regenerating nerves, determined on an amino acid analyzer, was found to be primarily spermidine and an unknown compound that migrated as a frontal elution peak. Autoradiographic analysis showed that the radioactivity was largely confined to axons, but a significant amount of the silver grains was associated with Schwann cells and myelin sheaths surrounding labeled axons in both intact and regenerating nerves. The data indicate that polyamine derivatives of putrescine are transported axonally in rat sciatic nerves, and some of this transported material accumulates in Schwann cells surrounding the labeled axons. These processes are apparently augmented during regeneration of the injured axons.     

Polyamines and Paraquat Toxicity in Arabidopsis thaliana

The relationship between paraquat toxicity and polyamine levelswas analyzed in Arabidopsis wild-type and gi-3 plants. Paraquattreatment led to an increase in putrescine, but not of spermidineand spermine content. Additionally, polyamine feeding offeredhigh levels of protection against paraquat toxicity with spermidinebeing the most effective protectant. (Received March 24, 1998; Accepted June 17, 1998)     

Transport of Abscisic Acid-2-C-14 in Intact Pea Seedlings

When abscisic acid-2-C-14 (AbA-2-C-14), 1 μg in 5 μl 40% ethanol, is applied to the apical bud of light-grown pea seedlings, C-14 is translocated downwards only in very small amounts and does not enter the root. In contrast to this, C-14 from indoleacetic acid-C-14 (IAA-2-C-14) applied in the same manner is translocated to the root where it accumulates. When AbA-2-C-14 is injected to the stem tissue at the apical bud, more labelled material is transported downwards than after application to the surface. Application of AbA-2-C-14 to an expanded leaf results in considerable accumulation of C-14 in the growing apical parts and in the lateral roots.     

Evidence for Symplasmic Ion Transport in Maize Roots

Excised maize roots, placed in saturated water vapour to limitthe external ionic supply, continued to produce exudates attheir basal ends for at least 24 h. The mean rate of fluid exudationfrom roots in water vapour was about 28 per cent of the correspondingrate in ‘control’ roote placed in a solution containing0.1 mil CaCl₂ and 1 mM KC1. Moreover, the net fluxes (mean ±S.E.)of potassium and calcium ions into the exudate were reducedfrom (35.8±3.2) x 10^– and (4.37±0.39) xlO^–9 mole cm^–2 h^– for roots in solution to(10.9±0.6) x 10^–9 and (l.00±0.06)x 10^–9molecm^–2 h^–1 respectively for roots in vapour. It isconsidered that the observation of a prolonged exudation ofwater and ions from the roots placed in water vapour demonstratesthe existence of an alternative ionic supply within the roottissue itself and that this parallel route of ion transportto the exudate constitutes the cortical symplasmic pathway. Pre-treatment of the excised roots with 0.8 M mannitol beforeexudation studies in water vapour and solution led to a significantreduction in the rates of fluid and ion exudation which hadbeen observed in untreated roots under similar conditions. Itis concluded that the plas-molysis, induced by mannitol, disruptedthe symplasmic connections between root cells and that thisperturbation significantly reduced the operation of the symplasmicmode of ion transport into the exudate.     

Sodium and Potassium Compartmentation and Transport across the Roots of Intact Spergularia marina

The Na⁺ and K⁺ transport characteristics of Spergularia marina (L.) Griseb. were considered in order to compare the systems by which these two physiologically different cations are managed during initial acquisition and subsequent partitioning in midvegetative plants. Uptake of ²²Na⁺ and ⁴²K⁺ and redistribution of labels in pulse-chase studies were compared under steady state growth conditions or with the concentration of one of the ions elevated. At high external concentrations, the initial ⁴²K⁺ accumulation and transport to the shoot was associated with a small, rapidly exchanging, cellular compartment similar to that previously indicated for Na⁺ (D Lazof, JM Cheeseman 1986 Plant Physiol 81: 742-747). At 1 mol m⁻³, K⁺ was conducted to the shoot through a root compartment, the specific activity of which rose much more slowly than the rapidly exchanging compartment. After a lag of approximately 5 minutes, ⁴²K⁺ translocation approached a constant rate with a half-time of 14 minutes compared to 5 minutes for ²²Na⁺ or for ⁴²K⁺ at higher external levels. At all external levels, prolonged translocation of ⁴²K⁺ was measured when a 10 minute pulse was followed by an unlabeled chase, again suggesting a conducting compartment distinct from that for Na⁺. It is suggested that the K⁺ conducting compartment, possibly the `bulk cytoplasm,' is associated with the active K⁺ transport system generally found in higher plants.     

Transport of Indole-3-Acetic Acid during Gravitropism in Intact Maize Coleoptiles

We have investigated the transport of tritiated indole-3-acetic acid (IAA) in intact, red light-grown maize (Zea mays) coleoptiles during gravitropic induction and the subsequent development of curvature. This auxin is transported down the length of gravistimulated coleoptiles at a rate comparable to that in normal, upright plants. Transport is initially symmetrical across the coleoptile, but between 30 and 40 minutes after plants are turned horizontal a lateral redistribution of the IAA already present in the transport stream occurs. By 60 minutes after the beginning of the gravitropic stimulus, the ratio of tritiated tracer auxin in the lower half with respect to the upper half is approximately 2:1. The redistribution of growth that causes gravitropic curvature follows the IAA redistribution by 5 or 10 minutes at the minimum in most regions of the coleoptile. Immobilization of tracer auxin from the transport stream during gravitropism was not detectable in the most apical 10 millimeters. Previous reports have shown that in intact, red light-grown maize coleoptiles, endogenous auxin is limiting for growth, the tissue is linearly responsive to linearly increasing concentrations of small amounts of added auxin, and the lag time for the stimulation of straight growth by added IAA is approximately 8 or 9 minutes (TI Baskin, M Iino, PB Green, WR Briggs [1985] Plant Cell Environ 8: 595-603; TI Baskin, WR Briggs, M Iino [1986] Plant Physiol 81: 306-309). We conclude that redistribution of IAA in the transport stream occurs in maize coleoptiles during gravitropism, and is sufficient in degree and timing to be the immediate cause of gravitropic curvature.     

Cellular Cobalt Fluxes in Roots and Transport to the Shoots of Wheat Seedlings

Macklon, A. E. S. and Sim, A. 1987. Cellular cobalt fluxes inroots and transport to the shoots of wheat seedlings.—J.exp. Bot. 38: 1663–1677. From compartmental analysis of radioisotope elution measurements,concentrations and fluxes of cobalt were estimated for corticalcells of wheat (Triticum aestivum L. cv. Fenman) seedling roots,relative to a complete nutrient solution containing 20 mmolm³ Co^{2 +} . The water free space, Donnan free space and cytoplasmcontained little Co relative to the high levels accumulatedin the vacuole, resulting in time courses of Co uptake whichwere essentially linear in continuous light. In light/dark cyclesthere was almost complete inhibition of uptake during the darkperiods. Comparison of estimated concentrations of Co in cytoplasmand vacuole with values to be expected, if passive diffusionalone was operative, indicated that active transport was occurringin an outward direction at the plasmalemma and into the vacuoleat the tonoplast. Transport to the shoot, which was unaffectedby transpirational water flux through the plant, reached about10% of root absorbed Co, most of which was stored in the corticalcell vacuoles and removed from the transport pathway. Between0·02 and 20 mmol m ³ Co in external solution, uptakewas linearly related to concentration on a log/log plot, andthe proportion of transported Co was constant. The implicationsof these findings are discussed. Key words: Cobalt, transmembrane fluxes, transport.     

Proline Metabolism and Transport in Maize Seedlings at Low Water Potential

The growing zone of maize seedling primary roots accumulatesproline at low water potential. Endosperm removal and excisionof root tips rapidly decreased the proline pool and greatlyreduced proline accumulation in root tips at low water potential.Proline accumulation was not restored by exogenous amino acids.Labelling root tips with [¹⁴C]glutamate and [¹⁴C]proline showedthat the rate of proline utilization (oxidation and proteinsynthesis) exceeded the rate of biosynthesis by five-fold athigh and low water potentials. This explains the reduction inthe proline pool following root and endosperm excision and theinability to accumulate proline at low water potential. Theendosperm is therefore the source of the proline that accumulatesin the root tips of intact seedlings. Proline constituted 10% of the amino acids released from the endosperm. [¹⁴C]Prolinewas transported from the scutellum to other parts of the seedlingand reached the highest concentration in the root tip. Less[¹⁴C]proline was transported at low water potential but becauseof the lower rate of protein synthesis and oxidation, more accumulatedas proline in the root tip. Despite the low biosynthesis capacityof the roots, the extent of proline accumulation in relationto water potential is precisely controlled by transport andutilization rate.     

Characteristics of Root-to-Shoot Transport of Cytokinin 6-Benzylaminopurine in Intact Seedlings of Citrus aurantium

Transport of the cytokinin 6-benzylaminopurine-8-¹⁴C in the root and shoot of intact Citrus aurantium L. seedlings was studied by “replacing” the 0.5 cm root tip with the uptake solution. The cytokinin was transported basipetally in the root and was distributed in an acropetal direction in the stem and into the leaves. Kinetic analysis of the transport for periods of up to 96 h revealed a characteristic advancing front of the label along the axis of the seedling. The estimated velocity of transport of 6-benzylamino-purine-8-¹⁴C in various regions of the intact root was 2.6 to 5.1 mm/h. The transport of 6-benzylaminopurine was predominantly in the transpiration stream, in stelar tissues of the root. Conditions of high transpiration favored enhanced transport to the shoot and an overall greater accumulation of the label. The total accumulation of 6-benzylaminopurine in roots of intact seedlings after 48 h of transport was 354% of that in roots of shoot-less seedlings. Root girdling and treatment of the root with KCN did not reduce the basipetal transport of the label in the root and into the shoot. Radiochromalogram scanning of root extracts and analysis of the ethanol insoluble-NaOH soluble fraction revealed considerable metabolic changes in the translocated cytokinin. Only 51% of the radioactivity remained in the original 6-benzylaminopurine peak after 24 h of incubation. Two other, unidentified, metabolites were detected. It is suggested that all the factors that affect the ascent of sap are involved in the long-distance transport of cytokinins, and that the rate and mode of transport of cytokinins from the root system to the shoot may be a major factor in the expression of their physiological activity.     

Partitioning of Electrons between the Cytochrome and Alternative Pathways in Intact Roots

To test the hypothesis that the cytochrome pathway is not invariably saturated when the alternative pathway is engaged, we titrated root respiration of several species with KCN (an inhibitor of the cytochrome pathway), both in the absence and presence of an inhibitor of the alternative pathway (salicylhydroxamic acid, SHAM). The slopes of the resultant KCN [rho] plots ([rho]cyt) were then used to determine whether the cytochrome pathway was saturated in each species. The species used were Festuca ovina ssp. ovina L., Phaseolus vulgaris L., and six Poa species (Poa pratensis L., Poa compressa L., Poa trivialis L., Poa alpina L., Poa costiniana Vick., and Poa fawcettiae Vick.). Although the cytochrome pathway was saturated in a number of species (i.e. [rho]cyt values were 1.0), several others exhibited [rho]cyt values of less than 0.5. Alternative pathway capacity correlated negatively with [rho]cyt, with [rho]cyt values of less than 1.0 occurring in tissues in which the alternative pathway capacity was greater than 25 to 30% of total respiration. The species that did not show full engagement of the cytochrome pathway rarely exhibited SHAM inhibition in the absence of KCN. We conclude that this lack of SHAM inhibition is not due to a lack of alternative pathway engagement but rather to the diversion of electrons from the alternative pathway to the unsaturated cytochrome path following the addition of SHAM.     

Role of K+ and Cl- in Osmotic Adjustment in Roots and Hypocotyls of Intact Mung Bean Seedlings

It was confirmed that osmotic adjustment occurred in young intactmung bean (Vigna mungo (L.) Hepper) seedlings exposed to highosmotic pressure stress. Root growth was not affected by osmoticpressure of less than 200 mOsra in the external solution, althoughhypocotyl growth was conspicuously reduced. Under this moderateosmotic stress, intracellular K⁺ concentration, [K⁺]_i, increaseddramatically during the osmotic adjustment in all the regionsof the root, but the intracellular Cl^– concentration,[Cl^–]_i, increased only in the aged mature region of theroot (28–33 mm from the root tip). About a half of theintracellular osmotic pressure in the aged mature region ofthe root could be ascribed to the contributions of [K⁺]_i and[Cl^–]_i, but in the hypocotyl, [K⁺]_i only contributed slightlyto the osmotic adjustment. (Received June 18, 1986; Accepted August 26, 1986)