Correlation between cell enlargement in pea internode segments and decrease in the pH of the medium of incubation

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 ...

Section: Resultsmentioning

confidence: 99%

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

Evans

Vesper²

1980

“…auxin may stimulate hydrogen ion secretion causing a reduction in pH within the cell wall to a growth-promoting level (6). This suggestion has received strong support by recent reports that auxin does stimulate hydrogen ion secretion to an extent sufficient to lower cell wall pH to about 5.0, a level capable of causing maximal stimulation of elongation (1,10,11 by examining the properties and pH dependence of a number of cell wall bound glycosidases in Avena, especially /-galactosidase. They found that the enzyme is inhibited by substances that inhibit the promotion of elongation by acid, and they reported that auxin enhances S-galactosidase activity in coleoptile segments by 36% after a 60-min treatment period.…”

mentioning

confidence: 89%

mentioning

confidence: 91%

Evidence Against the Involvement of Galactosidase or Glucosidase in Auxin- or Acid-stimulated Growth

Evans¹

1974

Research on the mode of action of auxin in the promotion of growth has shown that auxin treatment leads to hydrogen ion secretion and wall acidification. It has recently been reported that auxin stimulates cell wail 8-galactosidase activity in Avena coleoptiles, presumably by causing cell wall acidification, since (2,3,5,6,12). This has led to the suggestion that the promotion of growth by auxin may be acidmediated, i.e. auxin may stimulate hydrogen ion secretion causing a reduction in pH within the cell wall to a growth-promoting level (6). This suggestion has received strong support by recent reports that auxin does stimulate hydrogen ion secretion to an extent sufficient to lower cell wall pH to about 5.0, a level capable of causing maximal stimulation of elongation (1,10,11 by examining the properties and pH dependence of a number of cell wall bound glycosidases in Avena, especially /-galactosidase. They found that the enzyme is inhibited by substances that inhibit the promotion of elongation by acid, and they reported that auxin enhances S-galactosidase activity in coleoptile segments by 36% after a 60-min treatment period. These data suggest the possibility that auxin stimulates growth by stimulating hydrogen ion leakage which activates cell wall glycosidases leading to wall loosening.If this model of auxin and acid action is correct, it should be possible to greatly reduce the growth response to auxin or acid using specific inhibitors of glycosidases. It is known that the aldonolactones of simple sugars are specific and potent inhibitors of glycosidases active on substrates composed of those sugars (9, and references cited therein). In the work reported here, the effect of galactonolactone and gluconolactone on in vivo /2-galactosidase and /3-glucosidase activity was measured. I found that these aldonolactones strongly inhibit ,B-galactosidase and /3-glucosidase activity but have no effect on growth promotion by auxin or acid.MATERIALS AND METHODS Plant Material. Oats (A vena sativa var. Victory) were planted in an arrangement similar to that of Wiegand and Schrank (14). They were placed under dim red light for 24 hr and then allowed to grow at room temperature in darkness for 48 hr. Coleoptiles about 3 cm long were selected for harvesting, and segments 10 mm long were cut from them beginning 3 mm from the tip. The leaf was removed from each segment, and the segments were used either for growth tests or Enzyme Assays. Enzyme assays were done as described by Johnson et al. (7). Twenty coleoptile segments were rinsed with distilled water and placed in a 25-ml Erlenmeyer flask containing 1.5 ml of 13 mM phosphate-citrate buffer with or without 0.1 mm IAA and allowed to incubate at 30 C for 30 min. After 30 min, the assay was initiated by adding 0.5 ml of 40 mM p-N-gal2 or p-N-gluc. To

“…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.…”

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

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.