Vanadate inhibition of auxin-enhanced H + secretion and elongation in pea epicotyls and oat coleoptiles

When abraded oat (Avenasativa L. cv Victory) leaf segments are floated on KCI solution, white Ught causes acidification of the solution external to leaf tissue. The presence of mannitol amp_les the light-induced proton secretion. Mature leaves as weO as young ones acidify the medium in lght, while senescing leaves (after 3 to 4 days incubated in water in the dark) lose the ability to produce this response to light. The decrease in Ho secretion is already measureable after as little as 30 minutes in darkness, while the increase in proteolysis rate was detected only after 6 hours in dark. The decrease in capacity to secrete protons is one of the symptoms of leaf senescence. Moreover, fusicoccin mimics light in stimulating H+ pumping and delaying the senescence in the dark. On the other hand vanadate, an apparent Inhibitor of plasma membrane H+ ATPase, blocks the acidification and promotes the chlorophyll and protein degradation in leaf segments during the 2-day period of incubation. These results, which show a parallel between cessation of H' secretion and acceleration of senescence, may suggest a regulatory role for H+ secretion in leaf senescence.According to the acid growth theory, HI' secretion is a prerequisite for auxin-induced growth during the growth phase of coleoptiles and etiolated seedling stems (5). Green leaf cells which usually do not respond to auxin, expand in response to white light (3,20). Recently, Van Volkenburgh and Cleland found that lightinduced growth in bean leaves is accompanied by light-induced proton excretion (21). These authors suggest that leaf cell enlargement may occur by the same mechanism, as in coleoptiles, but that in this case proton excretion is induced by the light rather than by auxin (21).Light is also very effective in regulating the last stage of the development of leaves, namely senescence. In the absence oflight, the rates of Chl and protein loss (the dominant features of leaf senescence) are accelerated. However, when the leaf segments are placed in light the whole syndrome of senescence is delayed (17).Evidence for the existence of a plasma membrane proton pump in providing driving force for ion transport and for the auxin function in cell wall loosening has been accumulated (16). However, the process of light-stimulated proton efflux in nonaquatic plants has so far been observed only in bean leaves and only during the cell enlargement stage of leaf development (21). The present report explores and characterizes the light-induced proton secretion by oat leaf segments. This paper will demonstrate that the cessation of H+ secretion is one of the earliest indicators of senescence. Moreover, the promoting effect of fusicoccin on the acidification in darkness and the concomitant delay of senescence might indicate a possible regulative role for the proton secretion during this last stage of development.MATERIALS AND METHODS Plant Material. Seeds of Avena sativa L. cv Victory were sown and grown on vermiculite for 7 d under continuous illumination (25 w/m2) at 25°0...

Section: Effect Of Light On H Secretion In Oat Leafmentioning

confidence: 98%

Light-Induced H⁺ Secretion and the Relation to Senescence of Oat Leaves

Gepstein¹

1982

“…This is shown, for instance, by the inhibition of IAA-mediated growth by inhibitors of plasma-membrane ATPase function (eg. orthovanadate; Jacobs and Taiz, 1980). However, whether the induction of proton excretion by IAA leads to an acidification of the cell wall that is sufficient to explain the simultaneously induced growth response depends on severa1 conditions that are not yet well known.…”

Section: Dlscusslonmentioning

confidence: 99%

Determination of Auxin-Dependent pH Changes in Coleoptile Cell Walls by a Null-Point Method

Schöpfer

1993

The present debate on the validity of the "acid-growth theory" of auxin (indole-3-acetic acid, IAA) action concentrates on the question of whether IAA-induced proton excretion into the cell wall is quantitatively sufficient to provide the shift in pH that is required to explain IAA-induced growth (see D.L. Rayle, R.E. Plant Physiol 99:1271-1274 for a recent apologetic review of the acid-growth theory). In the present paper a nullpoint method has been employed for determining the growtheffective cell-wall pH i n the presence and absence of IAA after 60 min of treatment. Elongation of abraded maize (Zea mays 1.) and oat (Avena sativa L.) coleoptile segments was measured with the high resolution of a displacement transducer. The abrasion method employed for rendering the outer epidermal cell wall permeable for buffer ions was checked with a dye-uptake method. Evidence is provided demonstrating that externally applied solutes rapidly and homogeneously penetrate into the epidermal wall, whereas penetration into the inner tissue walls is strongly retarded. "Titration" curves of IAA-induced and basal elongation were determined by measuring the promoting/inhibiting effect of medium pH under iso-osmotic conditions in the range of p H 4.5 to 6.0. In maize, the null point (no pH-dependent change i n elongation rate after 5-10 min of treatment with 10 mmol L-' citrate buffer) was p H 5.00 after 60 min of IAA-induced growth, and the null-point p H determined similarly in IAA-depleted tissue (10 times smaller elongation rate) was 5.25. Corresponding titration curves with Avena segments led to slightly lower null-point p H values both in the presence and absence of IAA-induced growth. After induction of acid-mediated extension by 1 pmol L-' fusicoccin (FC) in maize, the null-point p H shifted to 3.9. At 0.5 pmol 1-', FC induced the same elongation rate as IAA but a 9-fold larger rate of proton excretion. At 0.033 pmol 1-', FC induced the same rate of proton excretion as IAA but had no appreciable effect on elongation. The implications of these results against the background of recent attempts to revitalize the acid-growth theory of IAA action are discussed.

“…At higher concentrations (usually >50 .tM), vanadate has been shown to inhibit ATPase activities (8,13,26,35) and ion transport (8,9,16) in various plant tissues. However, at these concentrations, vanadate also inhibits plant and animal phosphatases (22,32,36).…”

mentioning

confidence: 99%

Effect of Vanadate, Molybdate, and Azide on Membrane-Associated ATPase and Soluble Phosphatase Activities of Corn Roots

Gallagher

Leonard²

1982

409

216

The effects of vanadate, molybdate, and azide on ATP phosphohydrolase (ATPase) and acid phosphatase activities of plasma membrane, mitochondrial, and soluble supernatant fractions from corn (Zea mays L. WF9 x M017) roots were investigated. Azide (0.1-10 miflimolar) was a selective inhibitor of pH 9.0-ATPase activity of the mitochondrial fraction, while molybdate (0.01-1.0 milUimolar) was a relatively selective inhibitor of acid phosphatase activity in the supernatant fraction. The pH 6.4-ATPase activity of the plasma membrane fraction was inhibited by vanadate (10-500 micromolar), but vanadate, at similar concentrations, also inhibited acid phosphatase activity. This result was confirmed for oat (Avena sativa L.) root and coleoptile tissues. While vanadate does not appear to be a selective inhibitor, it can be used in combination with molybdate and azide to distinguish the plasma membrane ATPase from mitochondrial ATPase or supernatant acid phosphatase.Vanadate appeared to be a noncompetitive inhibitor of the plasma membrane ATPase, and its effectiveness was increased by KV. K -stimulated ATPase activity was inhibited by 50% at about 21 micromolar vanadate. The rate of K+ transport in excised corn root segments was inhibited by 66% by 500 micromolar vanadate.fungal (3, 4) cells. At higher concentrations (usually >50 .tM), vanadate has been shown to inhibit ATPase activities (8,13,26,35) and ion transport (8, 9, 16) in various plant tissues. However, at these concentrations, vanadate also inhibits plant and animal phosphatases (22,32,36). Mitochondrial ATPase activity is not affected by up to 500 ,UM vanadate, but higher concentrations (1 mM) have been reported to inhibit respiration and oxidative phosphorylation in rat liver mitochondria (11). Vanadate is clearly not a specific inhibitor of plasma membrane-ATPase, but it may be useful if used in combination with other inhibitors.Mitochondrial (and/or chloroplast) ATPase, acid phosphatase from the vacuole, and other phosphatases which readily utilize ATP as a substrate have caused considerable problems in studies on plasma membrane ATPase (14). It seems that molybdate is an inhibitor of phosphatase (34,36), although the specificity of molybdate for phosphatase over ATPase has not been reported. Azide is among several potent inhibitors of mitochondrial type, F1-ATPases (4).In this paper, we report the results of an investigation designed to compare the effects of vanadate, molybdate, and azide on ATPase and phosphatase activities of plasma membrane, mitochondrial, and soluble supernatant fractions from corn roots. It seems that plasma membrane ATPase activity can be distinguished by its sensitivity to vanadate and insensitivity to molybdate or azide.Progress in elucidating the role of plasma membrane-associated ATP phosphohydrolase (ATPase) in ion transport has been limited by the lack of a specific inhibitor of the enzyme. The availability of such an inhibitor is important for studies seeking to correlate effects on ATPase activity with changes in ion tra...