Regulation of Apoplastic pH in Source Leaves of Vicia faba by Gibberellic Acid

Abstract

Leaf discs of broad bean (Vicia faba L.), peeled on the spongy mesophyll side, rapidly altered the pH of the surrounding medium (apoplast). Using pH indicator paper appressed against the leaf, immediately after peeling, initial apoplastic pH was estimated to be 4.5. Changes in the apoplastic pH were measured with a microelectrode placed into a 100-microliter drop of an unbuffered solution (2 millimolar KCI, 0.5 millimolar CaC12, and 200 millimolar mannitol) on the peeled surface. Discs acidified the medium until the pH stabilized at about 5.0 (about 10 minutes). Acidification was inhibited by 50 micromolar sodium vanadate, an inhibitor of the plasmalemma H'-ATPase and attenuated by omitting the osmoticum or potassium ions from the medium. Fusicoccin (10 micromolar) greatly enhanced the rate of acidification. The presence of 0.1 to 1 micromolar gibberellic acid resulted in a slower rate of medium acidification. Gibberellic acid appeared to modulate the activity of the H'-translocating ATPase located at the plasma membrane of the mesophyll cells.Extracellular pH plays various regulatory roles in several physiological processes in plants. For example, the acid-induced growth hypothesis relates auxin action in elongating coleoptiles to cell wall acidification (14) and cell expansion in bean leaves is related to proton secretion into the apoplast ( 17, 18). Furthermore, transport of amino acids, sugars and other solutes across the plasmalemma is known to be a H+-cotransport mechanism, a process which is highly dependent on the pH gradient across the plasma membrane (5,11,13,15,19). Accordingly, pH sensitivity of sucrose uptake in various species is interpreted to be consistent with the H+/sucrose cotransport mechanism operating in different tissues including phloem (3, 6, 7, and references therein).Recently, we reported (1) the stimulatory effect of GA3 on sucrose export out ofattached Viciafaba source leaves, indicating a role for GA3 in the regulation of phloem loading and translocation rates. Combining in vitro and in vivo expermimental approaches, Daie et al. (2) have also shown a direct effect of GA3 on sucrose loading in celery. Furthermore, a stimulatory effect of GA3 on the activity of proton-translocating ATPase was reported for isolated phloem of celery (4). These data collectively suggested that GA3-enhancement of phloem loading may be mediated through the modification of the H+ gradient across the plasmalemma.In the present study, we examined the effects of GA3 on the acidification of the external medium by Vicia faba leaf discs. The data suggest that GA3 regulates the apoplastic pH.MATERIALS AND METHODS Plant Material. Vicia faba L. (cv. Broadwinsor) plants were grown in the greenhouse in 3-L pots containing a 7:3 sand:peat mixture and were irrigated daily with a complete nutrient solution. Day and night temperatures were set at 25 and 18°C, respectively. A 16-h photoperiod was provided using incandescent and fluorescent lamps with photon flux density of approximately 500 ,umol m-2 s-' at the can...

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“…Acid-induced growth was the first finding to indicate a signalling function for protons in plants. Apoplastic pH seems to be regulated by various plant hormones, such as auxin, gibberellic acid, and abscisic acid (Aloni et al, 1988;Blatt and Armstrong, 1993). Apoplastic pH is, in most plants, around pH 5, with extremes ranging from pH 4 to pH 7 (Grignon and Sentenac, 1991).…”