Regulation of K+ Channels in Maize Roots by Water Stress and Abscisic Acid

Root cortical and stelar protoplasts were isolated from maize (Zea mays L.) plants that were either well watered or water stressed, and the patch-clamp technique was used to investigate their plasma membrane K⁺ channel activity. In the root cortex water stress did not significantly affect inward- or outward-rectifying K⁺ conductances relative to those observed in well-watered plants. In contrast, water stress significantly reduced the magnitude of the outward-rectifying K⁺ current in the root stele but had little effect on the inward-rectifying K⁺ current. Pretreating well-watered plants with abscisic acid also significantly affected K⁺ currents in a way that was consistent with abscisic acid mediating, at least in part, the response of roots to water stress. It is proposed that the K⁺ channels underlying the K⁺ currents in the root stelar cells represent pathways that allow K⁺ exchange between the root symplasm and xylem apoplast. It is suggested that the regulation of K⁺ channel activity in the root in response to water stress could be part of an important adaptation of the plant to survive drying soils.     

Temperature-Dependent Water and Ion Transport Properties of Barley and Sorghum Roots : II. Effects of Abscisic Acid

Water flux through excised roots (J_v) is determined by root hydraulic conductance (L_p) and the ion flux to the xylem (J_i) that generates an osmotic gradient to drive water movement. These properties of roots are strongly temperature dependent. Abscisic acid (ABA) can influence J_v by altering L_p, J_i, or both. The effects of root temperature on responses to ABA were determined in two species differing in their temperature tolerances. In excised barley (Hordeum vulgare L.) roots, J_v was maximum at 25°C; 10 micromolar ABA enhanced J_v, primarily by increasing L_p, at all temperatures tested (15-40°C). In sorghum (Sorghum bicolor L.) roots, J_v peaked at 35°C; ABA reduced this optimum temperature for J_v to 25°C by increasing L_p at low temperatures and severely inhibiting J_i (dominated by fluxes of K⁺ and NO₃⁻) at warm temperatures. The inhibition of K⁺ flux by ABA at high temperature was mostly independent of external K⁺ availability, implying an effect of ABA on ion release into the xylem. In sorghum, ABA enhanced water flux through roots at nonchilling low temperatures but at the expense of tolerance of warm temperatures. These effects imply that ABA may shift the thermal tolerance range of roots of this heat-tolerant species toward cooler temperatures.     

The Metabolism and Transport of Abscisic Acid During Grain Fill in Wheat

The metabolism and transport of (±)-l2-¹⁴Clabscisic acidand its metabolites was investigated during the period of graindevelopment in wheat. Forty-five hours after feeding the hormoneinto the flag leaf blade, or after injection into the grains,nine metabolites could be extracted with acetone. Four of thesecompounds have been identified. They are phaseic acid, dihydrophaseicacid, abscisyl-ß-D-glucopyranoside and the polar metabolite.As well as the acetone-soluble metabolites a number of othershave been found which are insoluble in acetone. These appearto be conjugated to lipids, ‘gluten-like’ proteins,and carbohydrates. ABA and its metabolites were transported to all parts of theplant above the flag leaf node when the radioactive hormonewas introduced into the blade. However, when it was injectedinto the grain the radioactivity remained there until the pointwhen dry matter accumulation ceased, after which it was foundonly in the peduncle. The results suggest that the increasing level of ABA duringthe period of dry matter accumulation is due to biosynthesiswithin the grain and that the decrease after dry matter accumulationceases is due to both metabolism and redistribution within theplant.     

Effect of Temperature on the Radial Exchange of Labelled Water in Maize Roots

The temperature dependence of the efflux kinetics of labelledwater in isolated maize roots has been studied. The purposeof these experiments was to obtain the energy of activation,E (kcal/mole), of the rate-limiting step in this radial exchangeprocess under various experimental conditions. Estimates ofE were obtained from linear relations between ln{D'_w} and thereciprocal of the absolute temperature; values of the apparentdiffusion coefficient, D'_w, of labelled water in the root werefound from an analytical treatment of the efflux data in termsof a cylindrical diffusion model. The energy of activation forlabelled-water exchange in normal roots was 14.9 kcal/mole.The corresponding value for ‘dead’ (boiled) rootswas 3.9 kcal/mole. These values of E substantiate the view thatin normal roots the penetration of water across the membranesof the root cells constitutes the rate-determining step in theefflux whereas in ‘dead’ roots extracellular diffusionof water is the source of rate-control. Similar temperature dependence studies were performed on theefflux kinetics from normal and ‘dead’ roots treatedwith 10^–5 M phenylmercuric acetate (PMA). The energiesof activation for labelled-water exchange in normal and ‘dead’roots under these conditions were respectively 15.5 and 5.3kcal/mole. Moreover, the results of the efflux experiments onPMA-treated roots were considered to indicate that this inhibitorproduces an alteration in some structural aspect of the rate-controlling‘membranes’.     

Proline Accumulation in Maize (Zea mays L.) Primary Roots at Low Water Potentials (I. Requirement for Increased Levels of Abscisic Acid)

We have characterized sulfate transport in the unicellular green alga Chlamydomonas reinhardtii during growth under sulfur-sufficient and sulfur-deficient conditions. Both the Vmax and the substrate concentration at which sulfate transport is half of the maximum velocity of the sulfate transport (K1/2) for uptake were altered in starved cells: the Vmax increased approximately 10-fold, and the K1/2 decreased approximately 7-fold. This suggests that sulfur-deprived C. reinhardtii cells synthesize a new, high-affinity sulfate transport system. This system accumulated rapidly; it was detected in cells within 1 h of sulfur deprivation and reached a maximum by 6 h. A second response to sulfur-limited growth, the production of arylsulfatase, was apparent only after 3 h of growth in sulfur-free medium. The enhancement of sulfate transport upon sulfur starvation was prevented by cycloheximide, but not by chloramphenicol, demonstrating that protein synthesis on 80S ribosomes was required for the development of the new, high-affinity system. The transport of sulfate into the cells occurred in both the light and the dark. Inhibition of ATP formation by the antibiotics carbonylcyanide m-chlorophenylhydrazone and gramicidin-S and inhibition of either F- or P-type ATPases by N,N-dicyclohexylcarbodiimide and vanadate completely abolished sulfate uptake. Furthermore, nigericin, a carboxylate ionophore that exchanges H+ for K+, inhibited transport in both the light and the dark. Finally, uptake in the dark was strongly inhibited by valinomycin. These results suggest that sulfate transport in C. reinhardtii is an energy-dependent process and that it may be driven by a proton gradient generated by a plasma membrane ATPase.     

Effects of Salinity on Water Transport of Excised Maize (Zea mays L.) Roots

The root pressure probe was used to determine the effects of salinity on the hydraulic properties of primary roots of maize (Zea mays L. cv Halamish). Maize seedlings were grown in nutrient solutions modified by additions of NaCl and/or extra CaCl₂ so that the seedlings received one of four treatments: Control, plus 100 millimolar NaCl, plus 10 millimolar CaCl₂, plus 100 millimolar NaCl plus 10 millimolar CaCl₂. The hydraulic conductivities (Lp_r) of primary root segments were determined by applying gradients of hydrostatic and osmotic pressure across the root cylinder. Exosmotic hydrostatic Lp_r for the different treatments were 2.8, 1.7, 2.8, and 3.4·10⁻⁷ meters per second per megapascals and the endosmotic hydrostatic Lp_r were 2.4, 1.5, 2.7, and 2.3·10⁻⁷ meters per second per megapascals, respectively. Exosmotic Lp_r of the osmotic experiments were 0.55, 0.38, 0.68, and 0.60·10⁻⁷ meters per second per megapascals and the endosmotic Lp_r were 0.53, 0.21, 0.56, and 0.54·10⁻⁷ meters per second per megapascals, respectively. The osmotic Lp_r was significantly smaller (4-5 times) than hydrostatic Lp_r. However, both hydrostatic and osmotic Lp_r experiments showed that salinization of the growth media at regular (0.5 millimolar) calcium levels decreased the Lp_r significantly (30-60%). Addition of extra calcium (10 millimolar) to the salinized media caused ameliorative effects on Lp_r. The low Lp_r values may partially explain the reduction in root growth rates caused by salinity. High calcium levels in the salinized media increased the relative availability of water needed for growth. The mean reflection coefficients of the roots using NaCl were between 0.64 and 0.73 and were not significantly different for the different treatments. The mean values of the root permeability coefficients to NaCl of the different treatments were between 2.2 and 3.5·10⁻⁹ meters per second and were significantly different only in one of four treatments. Cutting the roots successively from the tip and measuring the changes in the hydraulic resistance of the root as well as staining of root cross-sections obtained at various distances from the root tip revealed that salinized roots had mature xylem elements closer to the tip (5-10 millimeters) compared with the controls (30 millimeters). Our results demonstrate that salinity has adverse effects on water transport and that extra calcium can, in part, compensate for these effects.     

High-Frequency Regeneration by Abscisic Acid (ABA) from Petiole Callus of Jatropha curcas

Jatropha curcas L. is attaining worldwide interest as an important biofuel crop. Experiments were conducted to improve the prevailing micropropagation technique as well as to develop a new ex vitro rooting method for J. curcas plant regeneration. Regeneration and ex vitro rooting efficiency was enhanced by augmenting the culture medium with abscisic acid (ABA). Different concentrations of 6-benzylaminopurine (BAP) and indole-3-butyric acid (IBA) were tested for callus generation from both in vitro and in vivo explants (leaf and petiole) on Murashige and Skoog (MS) medium. The best regenerative callus was achieved on MS medium supplemented with BAP (4.44 μM) and IBA (2.45 μM) from in vitro-cultured petioles. Highest regeneration (91%) was achieved by culturing petiole callus on MS medium supplemented with BAP (8.88 μM), IBA (0.49 μM), and ABA (1.9 μM), whereas 61% regeneration was obtained from in vitro leaf callus. Shoot proliferation and elongation was achieved on BAP (2.22 μM) and IAA (8.56 μM) with 10–13 shoots per explants. Highest rooting (65%) was achieved from M1 shoots (BAP, IAA, and ABA) on MS medium supplemented with IBA (2.45 μM), naphthaleneacetic acid NAA (0.54 μM), and 0.02% activated charcoal. Ex vitro rooting of 1-mo-old M1 shoots obtained from the charcoal-containing medium resulted optimum rooting (>72%) when transferred to polybags containing sterile sand. The plantlets were successfully acclimatized in soil with more than 98% survival rate in the greenhouse.     

Some Structural Changes During Ripening of Mangoes (Mangifera indica var. Alphonso) by Abscisic Acid Treatment

Abscisic acid at 10^–6 M concentration enhances ripeningof mangoes. The cells in the pulp of the fruit are large andparenchymatous and lose their integrity due to cell wall hydrolysisat the ripe stage. They contain abundant polysaccharides, consistingmainly of starch, which is degraded during ripening. The chloroplastsare transformed to chromoplasts containing red or yellow carotenoidpigment. Abscisic acid treatment enhances all of these processes.Mitochondria, on the other hand, retain their structural integritythroughout the ripening process in untreated and abscisic acid-treatedmangoes. Mangoes, ripening, abscisic acid, structure     

Effects of Abscisic Acid and of Hydrostatic Pressure Gradient on Water Movement through Excised Sunflower Roots

The effect of abscisic acid on the exudation rate from decapitated roots of sunflower plants (Helianthus annuus L.) was investigated in the presence and absence of an imposed hydrostatic pressure gradient. The magnitude of the abscisic acid effect was constant even when suctions up to 60 cm Hg were applied to the cut stumps.     

Water Transport in Onion (Allium cepa L.) Roots (Changes of Axial and Radial Hydraulic Conductivities during Root Development)

The hydraulic architecture of developing onion (Allium cepa L. cv Calypso) roots grown hydroponically was determined by measuring axial and radial hydraulic conductivities (equal to inverse of specific hydraulic resistances). In the roots, Casparian bands and suberin lamellae develop in the endodermis and exodermis (equal to hypodermis). Using the root pressure probe, changes of hydraulic conductivities along the developing roots were analyzed with high resolution. Axial hydraulic conductivity (Lx) was also calculated from stained cross-sections according to Poiseuille's law. Near the base and the tip of the roots, measured and calculated Lx values were similar. However, at distances between 200 and 300 mm from the apex, measured values of Lx were smaller by more than 1 order of magnitude than those calculated, probably because of remaining cross walls between xylem vessel members. During development of root xylem, Lx increased by 3 orders of magnitude. In the apical 30 mm (tip region), axial resistance limited water transport, whereas in basal parts radial resistances (low radial hydraulic conductivity, Lpr) controlled the uptake. Because of the high axial hydraulic resistance in the tip region, this zone appeared to be "hydraulically isolated" from the rest of the root. Changes of the Lpr of the roots were determined by measuring the hydraulic conductance of roots of different length and referring these data to unit surface area. At distances between 30 and 150 mm from the root tip, Lpr was fairly constant (1.4 x 10-7 m s-1 MPa-1). In more basal root zones, Lpr was considerably smaller and varied between roots. The low contribution of basal zones to the overall water uptake indicated an influence of the exodermal Casparian bands and/or suberin lamellae in the endodermis or exodermis, which develop at distances larger than 50 to 60 mm from the root tip.     

Three Classes of Abscisic Acid (ABA)-Insensitive Mutations of Arabidopsis Define Genes that Control Overlapping Subsets of ABA Responses

Wild type and three abscisic acid (ABA)-insensitive mutants of Arabidopsis (ABI1, ABI2, and ABI3) were compared for their ability to respond to ABA for a variety of ABA-inducible responses throughout the life cycle of the plants. The responses tested included effects on seedling growth, proline accumulation in seedlings, ABA-regulated protein synthesis in plantlets, and seed storage protein and lipid synthesis and accumulation. The abi1 and abi2 mutants showed reduced sensitivity to ABA for inhibition of seedling growth, induction of proline accumulation, and alterations in protein synthesis patterns during vegetative growth, but had wild type levels of storage reserves. In contrast, the abi3 mutant had wild type sensitivity for induction of proline accumulation and was only slightly less responsive to ABA with respect to effects on seedling growth and changes in patterns of protein synthesis. The major effects of this mutation were on seed development. Seeds of the abi3 mutant had two-thirds of the wild type level of storage protein and one-third the wild type level of eicosenoic acid, the major fatty acid component of storage lipids in wild type seeds. These results show that none of the abi mutants is insensitive for all ABA-inducible responses and that the abi3 effects are not seed-specific. Comparison of the degree of ABA sensitivity of monogenic mutant lines with that of digenic mutant lines carrying pairwise combinations of the abi mutations suggests that ABA responses in mature seeds are controlled by at least two parallel pathways.     

Timing of Kernel Development in Water-stressed Maize: Water Potentials and Abscisic Acid Concentrations

Maize (Zea mays L. cv. Pioneer 3925) subjected to post-anthesiswater stress during the first 2 weeks of kernel developmenthad lower leaf-water potentials and higher leaf-ABA concentrationsthan well-watered controls. There was a concomitant rise inABA concentration in kernel tissues 3 and 7 d after pollination(DAP), after which the concentration decreased to control levelsby 13 DAP. Kernel water potential, however, remained unchangedby the water stress. Radiolabelled ABA, fed to a leaf, was translocatedto kernels, where free ABA as well as several ABA metaboliteswere the major labelled fractions. This suggested that the stress-inducedkernel ABA was of maternal origin. Since ABA plays a putativerole in seed maturation of several crop species, and appliedABA or water stress often hastens seed development, we expectedthat a water-stress-induced rise in kernel ABA concentrationearly in grain development may serve to prematurely induce storage-productaccumulation. Zein, starch and several enzymes key to the starchsynthesis pathway followed the same course of induction throughoutthe experiment, with no difference between treatments Henceit was concluded that although water stress increased kernelABA independent of kernel water status, there was no apparenteffect of water stress or ABA on timing of early kernel developmentalprocesses. Zea mays L. cv. Pioncer 3925, maize, water stress, abscisic acid, endosperm development     

Transport of Water and Solutes across Maize Roots Modified by Puncturing the Endodermis (Further Evidence for the Composite Transport Model of the Root)

The effects of puncturing the endodermis of young maize roots (Zea mays L.) on their transport properties were measured using the root pressure probe. Small holes with a diameter of 18 to 60 [mu]m were created 70 to 90 mm from the tips of the roots by pushing fine glass tubes radially into them. Such wounds injured about 10-2 to 10-3% of the total surface area of the endodermis, which, in these hydroponically grown roots, had developed a Casparian band but no suberin lamellae. The small injury to the endodermis caused the original root pressure, which varied from 0.08 to 0.19 MPa, to decrease rapidly (half-time = 10-100 s) and substantially to a new steady-state value between 0.02 and 0.07 MPa. The radial hydraulic conductivity (Lpr) of control (uninjured) roots determined using hydrostatic pressure gradients as driving forces was larger by a factor of 10 than that determined using osmotic gradients (averages: Lpr [hydrostatic] = 2.7 x 10-7 m s-1 MPa-1; Lpr [osmotic] = 2.2 x 10-8 m s-1 MPa-1; osmotic solute: NaCl). Puncturing the endodermis did not result in measurable increases in hydraulic conductivities measured by either method. Thus, the endodermis was not rate-limiting root Lpr: apparently the hydraulic resistance of roots was more evenly distributed over the entire root tissue. However, puncturing the endodermis did substantially change the reflection ([sigma]sr) and permeability (Psr) coefficients of roots for NaCl, indicating that the endodermis represented a considerable barrier to the flow of nutrient ions. Values of [sigma]sr decreased from 0.64 to 0.41 (average) and Psr increased by a factor of 2.6, i.e. from 3.8 x 10-9 to 10.1 x 10.-9 m s-1(average). The roots recovered from puncturing after a time and regained root pressure. Measurable increases in root pressure became apparent as soon as 0.5 to 1 h after puncturing, and original or higher root pressures were attained 1.5 to 20 h after injury. However, after recovery roots often did not maintain a stable root pressure, and no further osmotic experiments could be performed with them. The Casparian band of the endodermis is discontinuous at the root tip, where the endodermis has not yet matured, and at sites of developing lateral roots. Measurements of the cross-sectional area of the apoplasmic bypass at the root tip yielded an area of 0.031% of the total surface area of the endodermis. An additional 0.049% was associated with lateral root primordia. These areas are larger than the artificial bypasses created by wounding in this study and may provide pathways for a "natural bypass flow" of water and solutes across the intact root. If there were such a pathway, either in these areas or across the Casparian band itself, roots would have to be treated as a system composed of two parallel pathways (a cell-to-cell and an apoplasmic path). It is demonstrated that this "composite transport model of the root" allows integration of several transport properties of roots that are otherwise difficult to understand, namely (a) the differences between osmotic and hydrostatic water flow, (b) the dependence of root hydraulic resistance on the driving force or water flow across the root, and (c) low reflection coefficients of roots.     

The Effect of Abscisic Acid (ABA) on the Respiration and Uptake of Inorganic Phosphate by Potato Pith Discs

Abscisic acid (ABA) stimulates the oxygen uptake and inhibits the uptake of inorganic phosphate of discs of potato pith. It is assumed that ABA acts as an uncoupler of the oxydative phosphorylation in agreement with what was shown earlier for β inhibitor from resting potato peelings. The effect is probably not directly related to growth phenomena.     

Water Transport Properties of Roots and Root Cortical Cells in Proton- and Al-Stressed Maize Varieties

Root and root cell pressure-probe techniques were used to investigate the possible relationship between Al- or H+-induced alterations of the hydraulic conductivity of root cells (LPc) and whole-root water conductivity (LPr) in maize (Zea mays L.) plants. To distinguish between H+ and Al effects two varieties that differ in H+ and Al tolerance were assayed. Based on root elongation rates after 24 h in nutrient solution of pH 6.0, pH 4.5, or pH 4.5 plus 50 [mu]M Al, the variety Adour 250 was found to be H+-sensitive and Al-tolerant, whereas the variety BR 201 F was found to be H+-tolerant but Al-sensitive. No Al-induced decrease of root pressure and root cell turgor was observed in Al-sensitive BR 201 F, indicating that Al toxicity did not cause a general breakdown of membrane integrity and that ion pumping to the stele was maintained. Al reduced LPc more than LPr in Al-sensitive BR 201 F. Proton toxicity in Adour 250 affected LPr more than LPc. In this Al-tolerant variety LPc was increased by Al. Nevertheless, this positive effect on LPc did not render higher LPr values. In conclusion, there were no direct relationships between Al- or H+-induced decreases of LPr and the effects on LPc. To our knowledge, this is the first time that the influence of H+ and Al on root and root cell water relations has been directly measured by pressure-probe techniques.     

Sensitivity to Abscisic Acid and Osmoticum Changes During Embryogenesis of Alfalfa (Medicago sativa)

Developing embryos of alfalfa can be placed into nine stagesof development according to their morphological characteristics.The intact seeds do not germinate when removed from the podand placed on water until at least stage VI, and complete germinabilityis not achieved until the seeds are at the stage when they startto undergo maturation desiccation. Isolated embryos are germinableas early as stage III, and will germinate within 24 h on Murashigeand Skoog medium containing 3% sucrose. Osmoticum can inhibitembryo germination, but only proper osmotic conditions can maintaindevelopment in vitro; development does not occur at germination-inhibitingconcentrations of abscisic acid. The sensitivity to abscisicacid and osmoticum changes with stage of development. Early-stageembryos have the highest abscisic acid sensitivity and thisdeclines to the extent that mature dry embryos require a highconcentration (1?0 mol m^–3) to prevent their germination.Sensitivity of the embryos to osmoticum is maximum at stageVII of development. The combined inhibitory effect of abscisicacid and osmoticum on germination during development is greaterthan their individual effects. This combined effect is stage-dependent.Thus studies on the effects of abscisic acid and osmoticum onembryogenesis and associated synthetic events should be expectedto vary according to the sensitivity to these agents at differentstages of development. Key words: Medicago sativa, embryogenesis, abscisic acid, osmoticum, seed development     

Effect of Osmotic Stress on Abscisic Acid Efflux and Compartmentation in the Roots of Two Maize Lines Differing in Drought Susceptibility

Roots of two Zea mays L. lines (drought-resistant Polj 17, and drought-susceptible F-2) were exposed to osmotic stress induced by sorbitol (osmotic potential –1.0 MPa). The following parameters were determined in cortex cells: membrane permeability for abscisic acid (ABA), ABA fluxes across membranes, pH values and ABA content in cytoplasm and vacuole. Osmotic stress induced different distribution of ABA within cell compartments in the investigated lines. ABA transport in the F-2 line occurred according to the intracellular pH gradient and the anion trap concept. In Polj 17, however, osmotic stress did not cause any significant effect on pH gradient and compartmental ABA content, but had a stimulating effect on ABA efflux from cytoplasm to apoplast and than via xylem to the leaf. These findings indicate different mechanisms of ABA transport between the investigated lines in response to osmotic stress.