Enhancement of Wall Loosening and Elongation by Acid Solutions

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: Introductionmentioning

confidence: 50%

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

Jacobs

Ray²

1976

146

“…Since the distance between the transducer shaft and root tip was large (120 mm) and total displacement before resetting was small (2 mm) ment angle varied only between 00 and 1°so tha (14) Figure 3, acid-induced elongation was recorded for 160 min with no sign of a decline. Thus acid-H induced elongation in intact pea roots is much more long lived than in coleoptile or corn root segments in which growth rate begins to decline 15 to 30 min after initiation (6,13). Figure 3 To recorder also shows that the acid growth response in pea roots can be obtained repeatedly by alternating between pH 6.5 and 4 in the root medium (lower curve).…”

mentioning

confidence: 83%

A New Sensitive Root Auxanometer

Evans¹

1976

A new sensitive root auxanometer is described. The auxanometer represents an adaption of the position-sensor transducer method to measurement of intact root elongation and has the advantages of simplicity and high sensitivity. Experiments with the auxanometer show that auxin begins to inhibit intact pea root elongation within 10 minutes and continues to inhibit elongation for at least 1 hour following a 1-hour treatment with the hormone. Exposure of pea roots to pH 4 results in a 2-to 3-fold increase in elongation rate beginning about 1 minute after acid treatment. Acid-induced elongation continues at a steady rate for at least 160 minutes and can be reinitiated repeatedly by shifting between pH 4 and 6.5. Auxin inhibits acid-induced elongation whether given before or after acidification, and a transient exposure to auxin renders intact roots relatively insensitive to acid for at least 1 hour after withdrawal of the hormone.There have been numerous reports dealing with the interaction of acid and auxin in the regulation of stem tissue elongation (4,7,12 slightly smaller in diameter than the swollen seed and had a notch to one side through which the emerging radicle could grow. The trays were filled with Meyer's solution (11) to the bottom surface of the Plexiglas platform and the solution was aerated during the subsequent growth of the roots. Roots were allowed to grow at room temperature in the laboratory under normal room lighting for 3 to 6 days at which time straight roots from 3 to 4 cm long were selected for experimentation. Although light is reported to inhibit root elongation (16, 17), there was no obvious effect of darkening the room on root elongation in this system during these short term experiments so these initial experiments were carried out in the laboratory under room lighting.Measurement of Elongation. Root elongation was measured by adapting the position-sensor transducer method (5) of recording stem elongation so that it could be used with intact roots. The pea seedling was mounted (Fig. 1) in a Plexiglas platform (D) fixed to the side of a specially constructed growth chamber 450 ml in volume so that the root protruded through a 3-mm hole in the bottom of the holder. The tip of the root was positioned in a glass capillary (E) 1.5 mm high and 2 mm in diameter fixed to the end of a copper wire (F) which passed through the rotating arm of a Metripak model 33-04 angular position-sensor transducer (G) and extended beyond to a counterweight (H) of 102 mg. The glass capillary was fixed to the end of the copper wire with fingernail polish which was also used to coat the submerged portion of the copper wire. The position-sensor transducer was provided with a constant 25 v power input from a HewlettPackard model 6215A DC power supply while output from the transducer was recorded using a Sargent-Welch model SRLG recorder. The chamber was filled with half strength Meyer's solution to the bottom of the seedling platform (D). The solution was continuously oxygenated through a 21 gauge stainless st...

“…The resultant acidification of the cell wall is thought to lead to wall loosening and accelerated growth (5,8,13,14).…”

Section: Introductionmentioning

confidence: 99%

An Improved Method for Detecting Auxin-induced Hydrogen Ion Efflux from Corn Coleoptile Segments

Evans

Vesper²

1980

Conditions necessary to detect maximal auxin-induced H+ secretion using a macroelectrode have been investigated using corn coleoptile segments. Auxin-induced H+ secretion is strongly dependent upon oxygenation or aeration when the tissue to volume ratio is high. Cuticle disruption or removal is also necessary to detect substantial auxin-induced H' secretion. The auxin-induced decrease in pH of the external medium is stronger when the hormone is applied to tissue in which the cuticle has been disrupted with an abrasive than when the hormone is applied to tissue from which the cuticle and epidermis have been removed by peeling. The lower detectable acidification ofthe external medium when using peeled segments appears to be due in part to the leakage of buffers into the medium and in part to the removal of the auxin-sensitive epidermal cells.The sensitivity of corn coleoptile segments to auxin, as measured by H+ secretion, increases about 2-fold during the first 2 hours after excision. Tlhis change in apparent sensitivity to auxin as reflected by H' secretion is paralleled by a time-dependent change in the growth response to auxin. Under optimal conditions for detecting H+ efflux (oxygenation, abrasion, hormone application 2 hours after excision), the latent period in auxininduced H+ efflux (about 7 or 8 minutes) is only half as great as the latent period in auxin-induced growth (about 18 to 20 minutes). These observations are consistent with the acid growth hypothesis of auxin action.According to the acid growth hypothesis of auxin action, auxin enhances growth by stimulating the secretion of H+ ions from the cytoplasm into the cell wall. The resultant acidification of the cell wall is thought to lead to wall loosening and accelerated growth (5,8,13,14).If auxin-induced H+ secretion acts as a mediator of auxininduced growth, one would expect hormone-induced H+ efflux to precede hormone-induced acceleration of growth. The most commonly used method of measuring H+ efflux is to place a large number (e.g. 40) of 1-cm long tissue segments in a small volume (e.g. 5 ml) of stirred weak buffer and monitor the change in pH of the medium in the presence and absence of hormone (12,14). Since the cuticle is said to offer substantial resistance to the entry and exit of H+ ions (3), it is common practice to remove the cuticle either by peeling the tissue segments with a pair of fine-tipped forceps or abrading them with emery powder. Using this method, auxin-induced H+ efflux appears to begin about 20 min or more after application of hormone (1,12,14) long apparent latent period in auxin-induced H+ secretion seems to be due, at least in part, to the large volume of the incubation solution compared with the volume of the cell wall fluid in which H+ would normally be accumulating. When the volume of the external medium is minimized, shorter latent periods are observed. Cleland (2), using a flat-tipped electrode resting on a row of peeled Avena coleoptile sections, reported an average latent period of 14 min in induction ...