Stoichiometric Correlation of Malate Accumulation with Auxin-dependent K+-H+ Exchange and Growth in Avena Coleoptile Segments

146

A pH microelectrode has been used to investigate the auxin effect on free space pH and its correbtion with auxin-stimulated elongation in segments of pea (Pisum sativum) stem According to the currently popular acid secretion hypothesis of auxin action (2, 7), auxins stimulate growth by causing acidification of the cell walls, which at low pH undergo an increase in extensibility that leads to rapid cell enlargement. Several lines of evidence are consistent with this hypothesis (7,9,19,24), but published measurements of auxin-induced release of acid from tissue into an external bathing medium (3,10,11,18) have not shown that auxin stimulates H+ secretion quickly enough to account for the rapid response (17) of elongation to auxin. If the acid secretion theory is correct it must be demonstrable that following exposure to an auxin the pH in the cell wall space falls to an elongation-stimulating value by the time that observable elongation in response to auxin actually commences, i.e. within the latent period for auxin action on elongation. A pH microelectrode that permits this type of measurement (13) Arbor, Mich.) of exposed tip length of 5 Aim and tip diameter of 2 ,um. The reference electrode was a segment of glass tubing (80 x 1 mm) the end of which was drawn into a capillary with a tip diameter of a few ,um. This tube was filled with 3 M KCI, in which was immersed an AgCI-coated silver wire that connected to the reference terminal of the pH meter. The electrode was read using the mv mode of operation of the meter and was calibrated against phosphate-citrate buffers (50 mm in K-phosphate and Na-citrate) of pH 4 and 6 before and after each experiment.The relation between pH of a buffer solution and mv reading by the pH microelectrodes was checked for several microelectrodes with a series of solutions varying in pH from 4 to 7. Because this relation was always found to be linear over the pH range, mv readings from experiments were converted to pH values by linear interpolation using the mv values measured for the calibration buffers of pH 4 and 6.The sensitivity of a pH microelectrode varied, for different experiments, from 50 to 58 mv/pH unit, but it was always constant through a given experiment. Between the beginning and the end of a typical experiment, however, the absolute mv values recorded by the microelectrode for the two calibration buffers would often change by a few mv. This drift in value was always of the same magnitude and in the same direction for both calibration buffers. Thus, the sensitivity of the microelectrode did not drift. Although calibration of the microelectrode against buffers of known pH before and after each experiment allowed us to monitor microelectrode drift, we used only the results of the postexperiment calibration for conversion of microelectrode mv readings to pH values.

Section: Discussionmentioning

confidence: 92%

mentioning

confidence: 99%

Rapid Auxin-induced Decrease in Free Space pH and Its Relationship to Auxin-induced Growth in Maize and Pea

Jacobs

Ray²

1976

146

“…5 The authors proposing this interpretation do not discuss the relationships between cytosolic pH changes and the well known activity of FC (9,14,19,28) (and also auxin [13,30]) in increasing the cytoplasm-acidifying synthesis of malate from phosphoenolpyruvate and CO2. It seems, however, relevant to point out that if cytoplasm acidification were a primary effect of FC (or of auxin), and if one accepts the current view that malate synthesis from phosphoenolpyruvate and bicarbonate is stimulated by cytosol alkalinization and inhibited by acidification (7,28) as predicted by the biochemical pH-stat theory (5,25), then cytosol acidification by FC (and, possibly, by auxin) would depend on their primary effect on the group of reactions leading to malate accumulation.…”

Section: Discussionmentioning

confidence: 99%

Stimulation of Weak Acid Uptake and Increase in Cell Sap pH as Evidence for Fusicoccin- and K⁺-Induced Cytosol Alkalinization

Marré¹,

Romani²,

Bellando³

et al. 1986

In maize root segments fusicoccin induced a consistent increase in cell sap pH (taken as representative of vacuolar pH). This effect was markedly enhanced by the presence of K' in the medium, whereas in the absence of fusicoccin K' did not significantly influence cell sap pH. Treatment with a weak acid at 2 mm concentration inhibited the uptake of a different ('4C-labeled) weak acid fed at a lower concentration, thus suggesting that acidification of the cytoplasm inhibits weak acid uptake. Fusicoccin and K' increased the rate of uptake of 5,5-dimethyloxazolidine-2,4-dione, butyric acid, or isobutyric acid slightly when fed separately, strongly when fed in combination. The synergism between fusicoccin and K' in stimulating weak acid uptake was parallel to that observed for the stimulation of H' extrusion. Application of the weak acid distribution method to a condition of 'quasi-equilibrium' indicated that fusicoccin induces a cytosolic pH increase of about 0.14 unit. These results are interpreted as providing circumstantial evidence that fusicoccin-and K'-induced stimulation of H' extrusion led to an alkalinization of the cytosol, and that other early metabolic responses, such as an increase in malate level, are a consequence of the increase in cytosolic pH.Recent work emphasizes the importance ofunderstanding how intracellular pH is regulated, and how cytosolic pH changes can influence cell activities. In animal systems the effects of factors or conditions stimulating growth are often associated with changes ofintracellular pH consequent to the activation ofa H / Na+ antiport at the plasma membrane (4). In plants, the main H' translocating system seems to consist of an ATP-driven, electrogenic H+ pump operating at the plasmalemma, apparently influenced by auxin and other natural hormones, and strongly activated by FC2, a toxin closely mimicking various aspects of auxin action (18,19 In the present research we have tried to evaluate qualitatively the FC-induced cytosolic pH changes by measuring in maize root segments the effect of the toxin on the uptake rate and on the accumulation of the permeating weak acids DMO, BA, and IBA. The measurement of the rate of weak acid influx had already been utilized by Marigo et al. (17). This method is based on the assumption that the uptake of an acid passively permeating only in the uncharged form would be proportional to the concentration gradient of this form across the plasmalemma; thus an alkalinization of the cytosol would accelerate the uptake of the acid by increasing its dissociation at the cytoplasmic side ofthe membrane, while an acidification would act in the opposite direction.An objection to this method is that its results are essentially qualitative. But the determination of intracellular pH by measurement of weak acid distribution at equilibrium is difficult in compact tissues because of the time required for equilibration, and other methods (such as 3'P-NMR, intracellular pH indicators, microelectrodes) are either too insensitive, too prone to error,...

“…However, by the use of the IR gas analyzer, it was shown that further increases in CO2 concentration above 1.0%/o had no additional effect on the rate of CO2 fixation. Besides affecting photosynthesis, CO2 has also been shown to affect various metabolic pathways via the control of intracellular pH (10) and an anaplerotic synthesis of amino acids (16). The interrelationships among these effects and the effects of CO2 on the metabolism and the rate of ethylene production has still to be elucidated.…”

mentioning

confidence: 99%

Effects of Carbon Dioxide on Ethylene Production and Action in Intact Sunflower Plants

Dhawan¹,

Bassi²,

Spencer³

1981