Membrane potential changes during transport of hexoses in Lemna gibba G1

The membrane potential (pd) of duck weed (Lemna gibba G1) proved to be energy dependent. At high internal ATP levels of 74 to 105 nmol ATP g^-1 FW, pd was between -175 and -265 mV. At low ATP levels of 23 to 46 nmol ATP g^-1 FW, pd was low, about -90 to -120 mV at pH 5.7, but -180 mV at pH 8. Upon addition of glucose in the dark or by light energy the low pd recovered to the high values. The active component of the pd was depolarized by the addition of hexoses in the dark and in the light. Hexose-dependent depolarization of the pd (= pd) followed a saturation curve similar to active hexose influx kinetics. Depolarization of the pd recovered in the dark even in the presence of the hexoses and with a 10fold enhancement in the light. Depolarization and recovery could be repeated several times with the same cell. Glucose uptake caused a maximum depolarization of 133 mV, fructose uptake half that amount, sucrose had the same effect as glucose. During 3-O-methylglucose and 2-deoxyglucose uptake the depolarizing effect was only slightly lower. The pd remained unchanged in the presence of mannitol. The glucose dependent pd and especially the rate of pd recovery proved to be pH-dependent between pH 4 and pH 8. It was independent of the presence of 1 mM KCl. Although no pH could be measured in the incubation medium, these results can be best explained by a H⁺-hexose cotransport mechanism powered by active H⁺ extrusion at the plasmalemma.Abbreviations LD longday - SD shortday - pd membrane electropotential difference - pd maximum membrane potential depolarization - L light - D dark - FW fresh weight - d days of culture of Lemna gibba - 1X perfusing solution without sugar, see methods     

Electron transport across the plasmalemma of Lemna gibba G1

Lemna gibba L., grown in the presence or absence of Fe, reduced extracellular ferricyanide with a V _max of 3.09 mol · g^-1 fresh weight · h^-1 and a K _m of 115 M. However, Fe³⁺-ethylenediaminetetraacetic acid (EDTA) was reduced only after Fe-starvation. External electron acceptors such as ferricyanide, Fe³⁺-EDTA, 2,6-dichlorophenol indophenol or methylene blue induced a membrane depolarization of up to 100 mV, but electron donors such as ferrocyanide or NADH had no effect. Light or glucose enhanced ferricyanide reduction while the concomitant membrane depolarization was much smaller. Under anaerobic conditions, ferricyanide had no effect on electrical membrane potential difference (E_m). Ferricyanide reduction induced H⁺ and K⁺ release in a ratio of 1.16 H⁺+1 K⁺/2 e^- (in +Fe plants) and 1.28 H⁺+0.8 K⁺/2 e^- (in -Fe plants). Anion uptake was inhibited by ferricyanide reduction. It is concluded that the steady-state transfer of electrons and protons proceeds by separate mechanisms, by a redox system and by a H⁺-ATPase.Abbreviations E _m electrical membrane potential difference - EDTA ethylenediaminetetraacetic acid - DCPIP dichlorophenol indophenol - +Fe control plant - -Fe iron-deficient plant - FW fresh weight - H⁺ electrochemical proton gradient     

Active hexose uptake in Lemna gibba G1

Growth of autotrophically growing duck-weeds (Lemna gibba L., G1) was stimulated by sucrose. The rate of respiration increased when plants had been grown on sucrose (8.7 mol O₂ g^-1 fresh weight (FW) h^-1) and was reduced after growth without sucrose in the dark or under longday conditions (2.5 mol O₂ g^-1 FW h^-1). Photosynthesis was induced already by low light intensities (0.1 klx).Short-time application of glucose or sucrose stimulated respiration in proportion to the hexose uptake rate. Sucrose is probably not taken up as the disaccharide. The transported sugar species after addition of sucrose are its hexose moieties produced by the high activity of the cell wall invertase. Fructose stimulated to a lesser extent; mannitol induced no enhancement; 2-deoxyglucose slightly inhibited O₂ uptake. After mild carbon starvation of the plants the uptake of glucose and 3-O-methylglucose proceeded without any lag phase, with similar saturation kinetics in both cases. The initial uptake rate at substrate saturation was 2.6 mol glucose g^-1 FW h^-1 in the dark. Light stimulated hexose uptake by 2 to 3 times. The results show that Lemna gibba has an energy-dependent constitutive system for hexose uptake.Abbreviation FW fresh weight - LD long day - SD short day     

Phytochrome-mediated changes in the membrane potential of subepidermal cells of Lemna paucicostata 6746

Light-stimulated transmembrane potential changes have been measured continuously after implantation of microelectrodes into subepidermal cells of the short-day plant Lemna paucicostata 6746. Irradiation for 5 min with white or red light caused a transient hyperpolarization. These potential changes could be suppressed with 10^-6 M DCMU. Irradiation of DCMU-inhibited plants with far-red light for 5 min hyperpolarized the membrane potential, which thereafter was not changed by further far-red application. Consecutive red light irradiation for 5 min depolarized the membrane potential. The red/far-red reversibility of the potential changes (which could be repeated several times with a single plant) suggests the participation of phytochrome.Abbreviations EDTA ethylenediaminetetraacetate - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - P_r, (P_fr) red- (far-red-) absorbing form of phytochrome     

Phosphate uptake inLemna gibba G1: energetics and kinetics

Phosphate uptake was studied by determining [³²P]phosphate influx and by measurements of the electrical membrane potential in duckweed (Lemna gibba L.). Phosphate-induced membrane depolarization (E _m ) was controlled by the intracellular phosphate content, thus maximal E _m by 1 mM H₂PO ₄ ^- was up to 133 mV after 15d of phosphate starvation. The E _m was strongly dependent on the extracellular pH, with a sharp optimum at pH 5.7. It is suggested that phosphate uptake is energized by the electrochemical proton gradient, proceeding by a 2H⁺/H₂PO ₄ ^- contransport mechanism. This is supported also by the fusicoccin stimulation of phosphate influx. Kinetics of phosphate influx and of E _m , which represent mere plasmalemma transport, are best described by two Michaelis-Menten terms without any linear components.Abbreviations E _m electrical membrane potential difference - E _m phosphate-induced, maximal membrane depolarization - FW fresh weight     

Evidence for proton/sulfate cotransport and its kinetics inLemna gibba G1

Sulfate uptake into duckweed (Lemna gibba G1) was studied by means of [³⁵S]sulfate influx and measurements of electrical membrane potential. Uptake was strongly regulated by the intracellular content of soluble sulfate. At the onset of sulfate uptake the membrane potential was transiently depolarized. Fusicoccin stimulated uptake up to 165% of the control even at pH 8. It is suggested that sulfate uptake is energized in the whole pH range by a 3H⁺/sulfate cotransport mechanism. Kinetics of sulfate uptake and sulfate-induced membrane depolarization in the concentration range of 5 M to 1 mM sulfate at pH 5.7 was best described by two Michaelis-Menten terms without any linear component. The second system had a lower affinity for sulfate and was fully active only at sufficiently high proton concentrations.Abbreviations c _o extracellular sulfate concentration - c _i intracellular sulfate concentration - E _m electrical membrane potential difference - E _m sulfate-induced, maximal membrane depolarization - electrochemical proton gradient - FW fresh weight     

pH and membrane-potential changes during glucose uptake inLemna gibba G1 and their response to light

Extracellular pH was measured with a microelectrode positioned over the lower surface of singleLemna gibba plants. Upon addition of glucose, a transient extracellular alkalinization occurred. Saturated extracellular pH changes were observed with 5 mM glucose. Simultaneously, the membrane potential difference of –250 mV in the dark measured with intracellular glass micropipettes, trnasiently decreased by 105 mV. Uptake of [¹⁴C]glucose and extracellular alkalinization was enhanced by light whereas glucose-induced membrane-potential changes were reduced in the light and became even smaller with increasing the preillumination time. Glucose uptake was optimal at pH 6. The results are taken as further evidence in favor of H⁺-glucose cotransport inLemna.Dedicated to Professor W. Simonis on the occasion of his 70th birthdayUniversity of Missouri Agricultural Experiment Station Journal Series, paper No. 8266     

Effect of abscisic acid on CO2 exchange in Lemna gibba

The effects of abscisic acid (ABA) on photosynthesis, dark respiration, and photorespiration were studied in Lemna gibba L. plants. The initial concentration of ABA in the nutrient solution was 10⁻⁷M and in a few experiments, 10⁻⁶M. The cultures were grown in the same solution for time periods ranging from one hour to 12 days. Net photosynthesis, measured as CO₂ uptake by infrared gas analyser technique, was inhibited after four hours of ABA treatment and reached a minimum after four to seven days depending on the time of the year. After 12 days a substantial recovery of photosynthesis was observed. Dark respiration was significantly stimulated after two to seven days of ABA treatment but then returned to the control level. The transient effects of ABA on photosynthesis and dark respiration corresponded to the previously measured time course of [¹⁴C]-ABA uptake by Lemna . Photorespiration measured as oxygen inhibition of photosynthesis was not affected by ABA.     

Amino acid uptake by Lemna gibba by a mechanism with affinity to neutral L-and D-amino acids

Earlier work suggested that amino acid uptake by Lemna gibba cells is a H⁺-cotransport mechanism driven by a proton-electrochemical gradient at the plasmalemma. The present investigations of the transient membrane depolarizations elicited by amino acids and tracer-uptake experiments show that all neutral -L-amino acids, D-alanine and analogues, like -alanine and p-fluorophenylalanine, are transported by the same system. It remains to be seen if there are separate mechanisms for the uptake of acidic and basic amino acids.     

Effect of abscisic acid on the transport of assimilates in barley

The effect of abscisic acid (ABA) on assimilate transport in barley was investigated in two parallel experiments. First, the effect upon [¹⁴C]sucrose transport from the flag leaf to the ear of a single ABA application made at different stages of growth of the fruits was investigated; the effect was measured 24 h after treatment. Second, the effect of a single application of ABA made at the same stages of growth as above on grain weight of the mature plant was investigated. In both types of experiments ABA was applied once to the ear of different plants as an aqueous solution (10^-3–10^-5 M), one to five weeks after anthesis. [¹⁴C] sucrose was applied by means of agar blocks. Parallel to these experiments, the endogenous content of ABA was investigated in the developing grains. When ears were treated with ABA two or four weeks after anthesis, an increase of up to 70% in the ¹⁴C-transport from the flag leaf to the ear was observed within a 24-h period after treatment (short duration experiments). At these growth stages the endogenous concentrations of ABA were low. In sharp contrast, ABA, especially in a concentration of 10^-3 M, decreased ¹⁴C-import from the flag leaf when applied three weeks after anthesis. At this stage the endogenous ABA content had reached its maximum. Long duration experiments with a single application of ABA to the car two weeks after anthesis resulted in a marked increase of weight per thousand kernels. ABA applications made earlier or later than two weeks after anthesis either reduced the grain weight or had no effect. It is concluded that ABA is involved in the regulation of assimilate transport from the leaves to the grains, possibly by influencing the unloading of sieve tubes in the ears. Promotion or inhibition of assimilate import by exogenously applied ABA may depend on the developmental stage of the grains and on the endogenous ABA level.Abbreviations ABA abscisic acid - TKW weight per thousand kernels     

Evidence for electrogenic proton extrusion by subepidermal cells of Lemna paucicostata 6746

The cell potential of Lemna paucicostata 6746 was measured between the vacuole and the external solution. The potential in the dark (-202 mV) could be depolarized with 0.1 mM dicyclohexyl carbodiimide (DCCD) or 1 mM arsenate to-81 mV. The hyperpolarization above the latter value is therefore attributed to an ATP-dependent process. The cell potential showed a significant dependence upon the pH of the external solution. The change in the potential induced by a jump in pH between two certain values, was reversible and independent of the mode of performing the pH change (stepwise or at once). The DCCD-or arsenate-depolarized potential did not respond to external pH changes. A 0.1 mM ammonium chloride solution depolarized the cell potential reversibly to-83 mV. This potential-change could be greatly reduced by simultaneous addition of 5 mM Na isobutyrate. The pH sensitivity of the cell potential is ascribed to changes in the rate of proton extrusion upon altering the proton gradient across the plasmalemma. The effects of ammonium and isobutyrate are interpreted as being the consequence of pH shifts at the inner face of the plasmalemma, caused by the permeation of the undissociated form of the weak acid or base. A critical discussion of an alternative interpretation for the ammonium effect is presented.Abbreviation DCCD N,N-dicyclohexyl carbodiimide     

Energy coupling for membrane hyperpolarization in Lemna: Evidence against an ATP-fueled electrogenic pump as the exclusive mechanism

The vacuolar electrical potential of Lemna paucicostata 6746 has an active component of about-130 mV. This hyperpolarization above the diffusion potential was maintained when dicyclohexyl carbodiimide (DCCD) or arsenate (0.1 mM or 5 mM final concentrations, respectively) were added in the light or after the plants had been kept in darkness for 1 h. The ATP level was reduced to 11±3% by DCCD and to 56±6% by arsenate under conditions identical to those during the potential measurements. In this report, it is discussed whether these results could be interpreted in terms of a putative electrogenic ATPase in the plasma membrane of Lemna. Rb⁺-influx in illuminated plants was 12.5% or 52% of the control when ATP generation was inhibited by DCCD or arsenate. This finding is regarded as justifying the assumption that the availability of ATP at plasmalemma-located transport sites is drastically decreased by these inhibitors.A passive proton-permeability in the cell membrane was induced with different concentrations of carbonyl cyanide m-chlorophenyl hydrazone (CCCP). The potential decrease, caused by the current through this shunt, was not affected by DCCD. It therefore seems less conceivable that the cell membrane remains hyperpolarized because of an increase of membrane resistance concomitant to the inhibition of the pump.The significance of respiratory processes for membrane hyperpolarization is displayed by the depolarizing action of anoxia or KCN. As ATP was found to be non-limiting under these conditions, the inhibition of the electrogenic pump is regarded as being in discord with the concept of an electrogenic ATPase, which is solely responsible for membrane hyperpolarization.Abbreviations CCCP carbonyl cyanide m-chlorophenyl hydrazone - DCCD N, N-dicyclohexyl carbodiimide - DES diethylstilbestro - DNP 2,4-dinitrophenol - POPOP 1,4-bis (2-(5-phenyloxazolyl))-benzene - PPO 2,5-diphenyloxazole     

Effect of indoleacetic acid,abscisic acid,root tips and coleoptile tips on growth and curvature of maize roots

The effect of externally applied indoleacetic acid (IAA) and abscisic acid (ABA) on the growth of roots of Zea mays L. was measured. Donor blocks of agar with IAA or ABA were placed laterally on the roots and root curvature was measured. When IAA was applied to vertical roots, a curvature directed toward the donor block was observed. This curvature corresponded to a growth inhibition at the side of the root where the donor was applied. When IAA was applied to horizontal roots from the upper side, normal geotropic downward bending was delayed or totally inhibited. The extent of retardation and the inhibition of curvature were found to depend on the concentration of IAA in the donor block. ABA neither induced curvature in vertical roots nor inhibited geotropic curvature in horizontal roots; thus the growth of roots was not inhibited by ABA. However, when, instead of donor blocks, root tips or coleoptile tips were placed onto vertical roots, a curvature of the roots was observed.Abbreviations ABA abscisic acid - IAA 3-indoleacetic acid     

Nickel and the metabolism of urea by Lemna paucicostata Hegelm. 6746

With urea as sole nitrogen source, the addition of 5×10^-5 M nickel sulfate to axenic cultures of Lemna paucicostata 6746 approximately doubles the rate of vegetative growth. Under a standard light-dark schedule, Ni²⁺ changes the daily pattern of respiratory CO₂ output on urea from one having a single daily peak to one with two daily peaks which resembles that on ammonium or nitrate as sole nitrogen source. It also increases CO₂ output by as much as 3-fold on a fresh-weight basis. These data represent the first confirmation in an intact higher plant of proposals, based on enzymology and tissue culture responses, for a role of Ni²⁺ in urea metabolism. Further, they indicate the possible existence of two distinct pathways of urea utilization.     

Effect of auxin and abscisic acid on cell wall extensibility in maize coleoptiles

Plastic and elastic in-vitro extensibilities (E _pland E _el ) of cell walls from growing maize (Zea mays L.) coleoptile segments were measured by stretching frozen-thawed tissue, pre-extended to its in-vivo length, at constant force (creep test) in a custom-buildt extensiometer, equipped with a linear-displacement transducer. The indole-3-acetic acid (IAA)-induced change of E _pl (E _pl ) is strictly correlated with the growth rate for a period of 3–4 h. Subsequently, E _plremains constant while the growth rate is slowing down. Since this discrepancy can be accounted for by a growth-dependent reduction of osmotic pressure, it is concluded that E _plrepresents quantitatively the relative increase of in-vivo extensibility (cell wall loosening) involved in IAA-mediated cell growth over a much longer time. On the other side it is argued that the growth rate may not be strictly correlated with wall extensibility during long-term growth. Abscisic acid (ABA) inhibits segment growth induced by auxin, fusicoccin, or exogenous acid, and this effect can be quantitatively attributed to an ABA-mediated reduction of cell wall extensibility as determined by the E _plmeasurement. Both, IAA and ABA have no effect on total protein synthesis, RNA synthesis, and amount of osmotic solutes. Fusicoccin-induced proton excretion is only slightly inhibited by ABA. In contrast to ABA, growth inhibition by cycloheximide (CHI) is always much larger than the concomitant reduction of E _pl , indicating that a further growth parameter is also involved in the inhibition of cell growth by CHI. E _el is not affected by either IAA, ABA, or CHI. It is concluded that E _pl as determined by the applied method, represents a relative measure of the actual in-vivo extensibility of the growing cell wall at the very moment when the tissue is killed, rather than an average extensibility accumulated over some immediate-past period of time as suggested by Cleland (1984, Planta 160, 514–520). Hence, we further draw the conclusion that IAA and ABA control of cell growth can entirely be attributed to a modulation of cell wall extensibility by these hormones in maize coleoptiles.Abbreviations ABA ±abscisic acid - CHI cycloheximide - E _el , E_pl elastic and plastic in vitro extensibilities, respectively (E _el+E_pl=E_tot>) - FC fusicoccin - IAA indole-3-acetic acid     

Radioimmunoassay for the determination of free and conjugated abscisic acid

The characterization and application of a radioimmunoassay specific for free and conjugated abscisic acid (ABA) is reported. The antibodies produced against a bovine serum albumin-(±)-ABA conjugate have a high affinity for ABA (K_a=1.3x10⁹l mol^-1). Trans, trans-ABA and related compounds, such as xanthoxin, phaseic acid, dihydrophaseic acid, vomifoliol or violaxanthin do not interfere with the assay. The detection limit of this method is 0.25x10^-12 mol ABA, the measuring range extends to 20x10^-12 mol, and average recoveries are 103%. Because of the high specificity of this immunoassay, no extract purification steps are required prior to analysis. Several hundred plants can be analyzed per day in a semi-automatic assay performance. ABA has been detected in all higher plant families examined, but was absent in the blue-green alga, Spirulina platensis, the liverwort Marchantia polymorpha, and two species of fungi.Abbreviations ABA abscisic acid - BHT 2.6-di-t-butyl-4-methyl phenol - TLC thin-layer chromatography - HSA human serum albumin Part 7 in the Series: Use of Immunoassay in Plant Science     

Short-term effects of plant hormones on membrane potential and membrane permeability of dwarf maize coleoptile cells (Zea mays L. d 1) in comparison with growth responses

The membrane potential difference of dwarf maize coleoptile cells is increased by both 10^-5moll^-1 gibberellic acid (GA₃) and indoleacetic acid (IAA) a few minutes after application. A final level is reached after 10–20 min. The membrane permeability ratio P _Na:P _K is altered by both hormones during the first 15 min after application, indicating a rapid effect on the membrane. Elongation growth of coleoptile segments, however, is only stimulated by IAA. The auxin-induced growth as well as the auxin effect on membrane permeability depends on the calcium ion concentration of the medium. It is concluded that IAA acts via a proton extrusion pump that is electrically balanced by a potassium ion uptake, driven by the electromotive force of the pump. The mode of action of GA₃ on elongation growth is assumed to involve a process that depends on the physiologic state of the tissue and/or metabolic energy.Abbreviations IAA indoleacetic acid - GA₃ gibberellic acid - FC fusicoccin - PD electric potential difference between the vacuole and the external medium     

Osmotic potential and abscisic acid regulate triacylglycerol synthesis in developing wheat embryos

This work was carried out to determine what factors in the developing wheat (Triticum aestivum L.) grain are involved in regulating the metabolism of the triacylglycerol (TAG) storage reserves. When embryos are isolated from the grain and incubated in media for 4 d the TAG content is affected in three ways. In the basal medium (dilute buffer) the content falls; in 30 mM sucrose the content remains unchanged; in sucrose supplemented with an osmoticum (400 mM mannitol) or abscisic acid (1 M ABA) the TAG content increases. Effective osmotic potentials and ABA concentrations fit well with their respective values in planta. The fatty-acid composition of TAG accumulated in vitro is close to that in planta but in the absence of ABA or osmoticum there is a fall in the C₁₈C₁₆ ratio. Experiments with [¹⁴C] acetate show that the in-planta rate of incorporation into TAG can only occur in isolated embryos treated osmotically or with ABA, while there seems to be no effect of these two factors on TAG breakdown. An osmotic shock (dilute buffer) for only 2 h causes a rapid fall in TAG synthesis which continues for ca. 24 h after which it recovers. Abscisic acid protects against osmotic shock. It is concluded that TAG synthesis in developing wheat embryos is regulated by the osmotic potential and-or ABA, and that the embryos are very sensitive to short-term perturbations of these two factors.Abbreviations ABA abscisic acid - dpa days post anthesis - TAG triacylglycerol We are grateful to the European Economic Community for a Fellowship to R.R.S. which provided financial support for this work.     

Effect of nitrogen stress and abscisic acid on nitrate absorption and transport in barley and tomato

The potential of barley (Hordeum vulgare L.) and tomato (Lycopersicon esculentum Mill.) roots for net NO ₃ ^- absorption increased two-to five fold within 2 d of being deprived of NO ₃ ^- supply. Nitrogen-starved barley roots continued to maintain a high potential for NO ₃ ^- absorption, whereas NO ₃ ^- absorption by tomato roots declined below control levels after 10 d of N starvation. When placed in a 0.2 mM NO ₃ ^- solution, roots of both species transported more NO ₃ ^- and total solutes to the xylem after 2 d of N starvation than did N-sufficient controls. However, replenishment of root NO ₃ ^- stores took precedence over NO ₃ ^- transport to the xylem. Consequently, as N stress became more severe, transport of NO ₃ ^- and total solutes to the xylem declined, relative to controls. Nitrogen stress caused an increase in hydraulic conductance (L _p) and exudate volume (J _v) in barley but decrased these parameters in tomato. Nitrogen stress had no significant effect upon abscisic acid (ABA) levels in roots of barley or flacca (a low-ABA mutant) tomato, but prevented an agerelated decline in ABA in wild-type tomato roots. Applied ABA had the same effect upon barley and upon the wild type and flacca tomatoes: L _p and J _v were increased, but NO ₃ ^- absorption and NO ₃ ^- flux to the xylem were either unaffected or sometimes inhibited. We conclude that ABA is not directly involved in the normal changes in NO ₃ ^- absorption and transport that occur with N stress in barley and tomato, because (1) the root ABA level was either unaffected by N stress (barley and flacca tomato) or changed, after the greatest changes in NO ₃ ^- absorption and transport and L _p had been observed (wild-type tomato); (2) changes in NO ₃ ^- absorption/transport characteristics either did not respond to applied ABA, or, if they did, they changed in the direction opposite to that predicted from changes in root ABA with N stress; and (3) the flacca tomato (which produces very little ABA in response to N stress) responded to N stress with very similar changes in NO ₃ ^- transport to those observed in the wild type.Abbreviation and symbols ABA abscisic acid - J_v exudate volume - L_p root hydraulic conductance     

Identification and measurement of indoleacetic and abscisic acids in the cambial region of Picea sitchensis (Bong.) Carr. by combined gas chromatography-mass spectrometry

Indole-3-acetic acid (IAA) and abscisic acid (ABA) were identified by combined gas chromatography-mass spectrometry (GCMS) in fractions obtained by diffusion and extraction from bark peelings of Sitka spruce. A procedure is described for the quantitative analysis of IAA and ABA levels in the same extract using the GCMS technique of single-ion current monitoring. This procedure was used to measure the diffusible, free, and bound fractions of IAA and ABA in the cambial region of Sitka spruce throughout one year; the range in concentration for these fractions was 0.06–0.30, 0.46–3.85, and 0.04–0.20 g/g oven-dry weight, respectively, for IAA, and 0–0.08, 0.03–2.21, and 0.13–0.66 g/g oven-dry weight, respectively, for ABA. Movement in the cambial region was found to be polar for endogenous IAA and nonpolar for endogenous ABA. Recoveries of [¹⁴C]IAA internal standards showed that 73–99.5% of the IAA was lost during purification, and that there could be up to 5-fold differences in recovery between purifications, indicating that IAA loss shold be measured in quantitative analyses.Abbreviations ABA aoscisic acid - GCMS combined gas chromatography-mass spectrometry - IAA indole-3-acetic acid - PVP polyvinylpyrrolidone - SICM single ion current monitoring - TMS trimethylsilyl