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Modification of the external pH by maize coleoptiles and velamen radicum of Vanilla planifoliaAndr .
I nsti tut fur Allgemeine Bot anik der Uni versitat Hamburg, Jun giusstraBe 6, D -2000 H ambur g 36, Federal Republ ic of Germany
Modification of the External pH by Maize Coleoptiles and Velamen radicum of Vanilla planijolio. ANDR. MICHAEL BOTTGER, H ANS-JURGEN So n and ANDREA GASCHE With 4 figures Received November 22,19 79 . Accepted January 23,19 80
Summary The rapid adjustment of the pH in the external bathing medium of maize coleoptiles is mainly due to the buffer capaci ty of the cell wall and to a simple physico-chemical process, as there is little difference between killed and living tissue. The adjustment occurs within 30 min with maize coleoptiles. Using Velamen radicum, a tissue highly specialized for water- and ion uptake the final pH is reached within one minute.
Key words: external pH , wall buf fer capacity, Zea mays, Vani lla planifo lia.
Introduction A still controversia l point of d iscussion is w et he r au xin induced st raight growth is med iated by proton-extrusion . Sever al investigators we re unable to detect afte r aux in-t rea tment a dramat ic auxi n-i nd uce d pH-drop. Others found a pH-shi ft in th e bathin g medium of segm ent s but al so in the co nt rol exper iments (PARRISH and D AVIES, 1977 ; VANDERHOEF er a l., 19 77). ILAN (1973) , BARLEY and LEOPOLD (1972 ), P ERLY et al ., 1975 , cha lleng ed th e aci d grow t h theory of auxi n ac ti on. U sin g microelectrod es, P ENNY et a1. (1975) observed no pH drop in the free sp ace , while LURSSEN (1978) d escribed a correlation between grow t h rat e and the pH of the surface of Avena coleoptiles. V ANDERHOEF et a1. (1977) suggested an active regulation of the external pH, as the fin al pH of 5.4 of the bathing medium was reached 60 minutes later and independentl y of proton concentration in th e beginning. In this case aux in had no effect on mod ific at ion of external pH by soyb ean hypocotyl cell s. But P ARRISH and D AVI ES (1977) sugg est ed that th e techn iqu es used for most ex tracellu la r pH studi es do not ve ry closely approxim ate in vivo conditions or p ro perl y intramu ral p roton co nce ntra tions. In our st udy we wa nt to d emonstra te th e influe nce of th e intramu ral pH on pH of the ex te rna l en vironment. Z. Pflanzenphysiol. Ed. 99. S. 89-93. 1980.
90
MICHAEL BOTTGER, HANS-]URGEN
SOLL
and ANDREA GASCHE
Materials and Methods Maize seeds were sown in moist vermiculite and cultivated in the dark at 24°C. After five days the cuticle of the coleoptiles was gently abraded with carborundum powder. Beginning 3 mm below the coleoptile tip, five mm long segments were cut. In some experiments, the coleoptile segments were killed by submersion in 20 Ofo methanol for 24 hours. Before the experiment they were rinsed with water and washed two times in H 20 bidest for 20 min each. Aerial roots of Vanilla planifolia ANDR., purchased by the Botanical Garden of Hamburg, were cut in 5 mm long segments and the velamen radicum was peeled and used for the experiment. Either 10 coleoptile segments or 0.5 g of the velamen radicum were used in each experiment. The plant materials were plunged in 3 mIlO mM NaCl and gently stirred at 22°C. Addition of NaCl was used in order to increase conductance for accurate pH measurement. The pH was either adjusted with 0.001 N HCl or NaOH and continously recorded with a Knick 624 pH-meter equipped with a Ingold 405-M8 electrode. The procedure was carried out at green safelight. The experiments were repeated at least three times.
Results and Discussion
As the cells are able to regulate their internal pH by a feed back mechanism and CLELAND, 1978 j STOUT et aI., 1978) we investigated whether cells may regulate the pH of the external bathing medium as well. For that, we changed the proton concentration at the beginning of each experiment and monitored the pH for one hour. Excised living maize coleoptile segments adjust the external hydrogen ion concentration to an pH of 5.6 ± 0.2 within 60 minutes (Fig. 1). This regulation is nearly independent of the pH at the beginning of the experiment. The adjustment is rather quick and half the difference between beginning and equilibrium pH was reached within ten minutes. (STOUT
8
pH
10
20
30
40
SO [Mifl
Fig. 1: Adjustment of the external pH of maize coleoptiles. At the beginning the pH of the 10 mM Na'Cl-solution was adjusted with HCl or NaOH. The final pH was reached within 30 minutes and was independent of the original pH. Z. Pflanzenphysiol. Bd. 99. S. 89-93. 1980.
Modification of the external pH
7
i'---~
91
~_
5-4-----------------
10
20
30
40
50 Min
Fig. 2: Control experiment with 10 mM Na Cl-solution and without plant-segments. A change of pH by atmospheric CO 2 can be only observed in the alkaline range.
The pH may be influenced by carbon dioxide generated by the plant segments or of atmospheric origin. Figure 2 shows, that simple dissolution of atmospheric CO 2 causes only a pH drop if the pH was initially alkaline. If the pH is initially acidic, no significant change could be observed as the solubility of CO 2 is strongly reduced at low pH. The adjustment of the external pH is assumed to be an active process (VANDERHOEF et al., 1977) by several proton pumps, some of them being inducible by auxin and fusicoccin (HAGER et al., 1971; CLELAND, 1975; MARRE et al., 1973). But the pectins and proteins of the cell wall may act as buffers and influence the pH of the external bathing medium. In order to determine the magnitude of this physicochemical process, we used in a second experiment methanol-killed coleoptiles.
ill
~
~
~
~~
Fig. 3: pH-changes caused by killed coleoptiles, The coleoptiles were killed by 20 % methanol for 24 hours and repeately washed in destilled water. Then the experiment was performed as described in fig. 1. Z. Pflanzenphysiol. Bd. 99. S. 89-93. 1980.
92
MICHAEL BOTTGER, HANS-JURGEN SOLL and ANDREA GASCHE
10
20
30
40
50
!i-1in]
Fig. 4: pH-changes caused by the dead cells of Velamen radicum. The experiment was performed as described in fig. 1. The final pH was reached within one minuts.
As Figure 3 demonstrates very few difference between living and dead tissue can be detected. The pH of the external environment seems to be regulated mainly by the high buffer capacity of the cell. The cell wall is mostly responsible for this adjustment as Velamen radicum of Vanilla shows the same final pH. This plant tissue has no protoplast because it dies during differentiation. The adjustment occurs completely within one minute as this tissue is specialized for water and ion uptake. The rapid pH adjustment of the environmental medium seems to be caused mainly by the high buffer capacity of the cell wall and is - in contrast to the conclusions of VANDERHOEF et al. (1977) - mostly an inactive process. Acknowledgements We thank Dr. BIEDERMANN for discussion. This work was supported by the DFG.
References BARKLEY, G. M. and A. C. LEOPOLD: Comparable effects of hydrogen ions, carbon dioxide, and auxin on pea stem segment elongation. - Plant Physio!. 52, 76-78 (1972). CLELAND, R. E.: Auxin-induced hydrogen ion excretion: correlation with growth and control by external pH and water stress. - Planta 127, 233-242 (1975). HAGER, A., H. MENZEL, and A. KRAUSS: Versuche und Hypothese zur Primarwirkung des Auxins beim Streckungswachstum. - Planta 100, 47-75 (1971). ILAN, I.: On auxin-induced pH drop and on the improbability of its involvement in the primary mechanism of auxin-induced growth promotion. - Physio!. Plant. 28, 146-118
(1973). LURSSEN, K.: The surface pH of Avena coleoptile and its correlation with growth rates. Plant Sci. Letters 13, 309-313 (1978). MARRE, E., P. LADO, F. R. CALDOGNO, and R. COLOMBO: Correlation between cell enlargement in pea internode segments and decrease in the pH of the medium of incubation. II. Z. Pflanzenphysiol. Bd. 99. S. 89-93. 1980.
Modi ficat ion of th e extern al pH
93
Effects of inhibitor s of respirat ion ox idati ve phosphorylation and protein synthesis. Plant Sci. Lett. 1, 185-192 (1973). PARRISH, D. J. and P. J. D AVI ES: On the relati ons hip between ext ra- cellular pH and th e grow th of excised pe a stem segments. - Plant Ph ysio!. 59, 574-678 (1977). PENNY, P., J. D UNLOP, J. E. PERLEY, and D. PENNY: pH an d au xin-induced growth : a causa l rela tion ship ? - Pl ant Sci. Lett. 4, 35- 40 (1975). PERLEY, J. E., D . P ENNY, an d P. P ENNY: A di fferen ce between auxin-ind uced and hydrogen ion induced growth. - Plant Sci. Lett . 4, 133- 136 (1975). STOUT, R. G. and R . E. CLELAND : Effects of fusicoccin on th e activi ty of a key pH-stat enzyme, PEP-Carboxylase. - Pl ant a 139, 43--45 (1978). STOUT, R. G., K. D . JOHNSON, and D . L. RAYLE : Rap id aux in- and fusicoccin-enhanced Rb " uptake and mal at e sy nt hesis in Av ena coleo pt ile sect io ns. - Planta 139, 35-41 (1978). VANDERHOEF, L. N., J. S. FINDLEY, J. J. BURKE, and W. E. BLiZZWARD: Aux in has no eff ect on modification of ex te rn al pH by soybean hypocotyl cells. - Plant Physiol , 59, 1000-1003 (1977).
M. BOTTGER, Institut fur All gemeine Botanik der Universitar Hamburg, jungiusstrafle 6, D-2000 Hamburg 36.
Z. Pflanzenph ysiol. Rd. 99. S. 89-93. 1980.
Modification of the External pH by Maize Coleoptiles and Velamen radicum of Vanilla planijolio. ANDR. MICHAEL BOTTGER, H ANS-JURGEN So n and ANDREA GASCHE With 4 figures Received November 22,19 79 . Accepted January 23,19 80
Summary The rapid adjustment of the pH in the external bathing medium of maize coleoptiles is mainly due to the buffer capaci ty of the cell wall and to a simple physico-chemical process, as there is little difference between killed and living tissue. The adjustment occurs within 30 min with maize coleoptiles. Using Velamen radicum, a tissue highly specialized for water- and ion uptake the final pH is reached within one minute.
Key words: external pH , wall buf fer capacity, Zea mays, Vani lla planifo lia.
Introduction A still controversia l point of d iscussion is w et he r au xin induced st raight growth is med iated by proton-extrusion . Sever al investigators we re unable to detect afte r aux in-t rea tment a dramat ic auxi n-i nd uce d pH-drop. Others found a pH-shi ft in th e bathin g medium of segm ent s but al so in the co nt rol exper iments (PARRISH and D AVIES, 1977 ; VANDERHOEF er a l., 19 77). ILAN (1973) , BARLEY and LEOPOLD (1972 ), P ERLY et al ., 1975 , cha lleng ed th e aci d grow t h theory of auxi n ac ti on. U sin g microelectrod es, P ENNY et a1. (1975) observed no pH drop in the free sp ace , while LURSSEN (1978) d escribed a correlation between grow t h rat e and the pH of the surface of Avena coleoptiles. V ANDERHOEF et a1. (1977) suggested an active regulation of the external pH, as the fin al pH of 5.4 of the bathing medium was reached 60 minutes later and independentl y of proton concentration in th e beginning. In this case aux in had no effect on mod ific at ion of external pH by soyb ean hypocotyl cell s. But P ARRISH and D AVI ES (1977) sugg est ed that th e techn iqu es used for most ex tracellu la r pH studi es do not ve ry closely approxim ate in vivo conditions or p ro perl y intramu ral p roton co nce ntra tions. In our st udy we wa nt to d emonstra te th e influe nce of th e intramu ral pH on pH of the ex te rna l en vironment. Z. Pflanzenphysiol. Ed. 99. S. 89-93. 1980.
90
MICHAEL BOTTGER, HANS-]URGEN
SOLL
and ANDREA GASCHE
Materials and Methods Maize seeds were sown in moist vermiculite and cultivated in the dark at 24°C. After five days the cuticle of the coleoptiles was gently abraded with carborundum powder. Beginning 3 mm below the coleoptile tip, five mm long segments were cut. In some experiments, the coleoptile segments were killed by submersion in 20 Ofo methanol for 24 hours. Before the experiment they were rinsed with water and washed two times in H 20 bidest for 20 min each. Aerial roots of Vanilla planifolia ANDR., purchased by the Botanical Garden of Hamburg, were cut in 5 mm long segments and the velamen radicum was peeled and used for the experiment. Either 10 coleoptile segments or 0.5 g of the velamen radicum were used in each experiment. The plant materials were plunged in 3 mIlO mM NaCl and gently stirred at 22°C. Addition of NaCl was used in order to increase conductance for accurate pH measurement. The pH was either adjusted with 0.001 N HCl or NaOH and continously recorded with a Knick 624 pH-meter equipped with a Ingold 405-M8 electrode. The procedure was carried out at green safelight. The experiments were repeated at least three times.
Results and Discussion
As the cells are able to regulate their internal pH by a feed back mechanism and CLELAND, 1978 j STOUT et aI., 1978) we investigated whether cells may regulate the pH of the external bathing medium as well. For that, we changed the proton concentration at the beginning of each experiment and monitored the pH for one hour. Excised living maize coleoptile segments adjust the external hydrogen ion concentration to an pH of 5.6 ± 0.2 within 60 minutes (Fig. 1). This regulation is nearly independent of the pH at the beginning of the experiment. The adjustment is rather quick and half the difference between beginning and equilibrium pH was reached within ten minutes. (STOUT
8
pH
10
20
30
40
SO [Mifl
Fig. 1: Adjustment of the external pH of maize coleoptiles. At the beginning the pH of the 10 mM Na'Cl-solution was adjusted with HCl or NaOH. The final pH was reached within 30 minutes and was independent of the original pH. Z. Pflanzenphysiol. Bd. 99. S. 89-93. 1980.
Modification of the external pH
7
i'---~
91
~_
5-4-----------------
10
20
30
40
50 Min
Fig. 2: Control experiment with 10 mM Na Cl-solution and without plant-segments. A change of pH by atmospheric CO 2 can be only observed in the alkaline range.
The pH may be influenced by carbon dioxide generated by the plant segments or of atmospheric origin. Figure 2 shows, that simple dissolution of atmospheric CO 2 causes only a pH drop if the pH was initially alkaline. If the pH is initially acidic, no significant change could be observed as the solubility of CO 2 is strongly reduced at low pH. The adjustment of the external pH is assumed to be an active process (VANDERHOEF et al., 1977) by several proton pumps, some of them being inducible by auxin and fusicoccin (HAGER et al., 1971; CLELAND, 1975; MARRE et al., 1973). But the pectins and proteins of the cell wall may act as buffers and influence the pH of the external bathing medium. In order to determine the magnitude of this physicochemical process, we used in a second experiment methanol-killed coleoptiles.
ill
~
~
~
~~
Fig. 3: pH-changes caused by killed coleoptiles, The coleoptiles were killed by 20 % methanol for 24 hours and repeately washed in destilled water. Then the experiment was performed as described in fig. 1. Z. Pflanzenphysiol. Bd. 99. S. 89-93. 1980.
92
MICHAEL BOTTGER, HANS-JURGEN SOLL and ANDREA GASCHE
10
20
30
40
50
!i-1in]
Fig. 4: pH-changes caused by the dead cells of Velamen radicum. The experiment was performed as described in fig. 1. The final pH was reached within one minuts.
As Figure 3 demonstrates very few difference between living and dead tissue can be detected. The pH of the external environment seems to be regulated mainly by the high buffer capacity of the cell. The cell wall is mostly responsible for this adjustment as Velamen radicum of Vanilla shows the same final pH. This plant tissue has no protoplast because it dies during differentiation. The adjustment occurs completely within one minute as this tissue is specialized for water and ion uptake. The rapid pH adjustment of the environmental medium seems to be caused mainly by the high buffer capacity of the cell wall and is - in contrast to the conclusions of VANDERHOEF et al. (1977) - mostly an inactive process. Acknowledgements We thank Dr. BIEDERMANN for discussion. This work was supported by the DFG.
References BARKLEY, G. M. and A. C. LEOPOLD: Comparable effects of hydrogen ions, carbon dioxide, and auxin on pea stem segment elongation. - Plant Physio!. 52, 76-78 (1972). CLELAND, R. E.: Auxin-induced hydrogen ion excretion: correlation with growth and control by external pH and water stress. - Planta 127, 233-242 (1975). HAGER, A., H. MENZEL, and A. KRAUSS: Versuche und Hypothese zur Primarwirkung des Auxins beim Streckungswachstum. - Planta 100, 47-75 (1971). ILAN, I.: On auxin-induced pH drop and on the improbability of its involvement in the primary mechanism of auxin-induced growth promotion. - Physio!. Plant. 28, 146-118
(1973). LURSSEN, K.: The surface pH of Avena coleoptile and its correlation with growth rates. Plant Sci. Letters 13, 309-313 (1978). MARRE, E., P. LADO, F. R. CALDOGNO, and R. COLOMBO: Correlation between cell enlargement in pea internode segments and decrease in the pH of the medium of incubation. II. Z. Pflanzenphysiol. Bd. 99. S. 89-93. 1980.
Modi ficat ion of th e extern al pH
93
Effects of inhibitor s of respirat ion ox idati ve phosphorylation and protein synthesis. Plant Sci. Lett. 1, 185-192 (1973). PARRISH, D. J. and P. J. D AVI ES: On the relati ons hip between ext ra- cellular pH and th e grow th of excised pe a stem segments. - Plant Ph ysio!. 59, 574-678 (1977). PENNY, P., J. D UNLOP, J. E. PERLEY, and D. PENNY: pH an d au xin-induced growth : a causa l rela tion ship ? - Pl ant Sci. Lett. 4, 35- 40 (1975). PERLEY, J. E., D . P ENNY, an d P. P ENNY: A di fferen ce between auxin-ind uced and hydrogen ion induced growth. - Plant Sci. Lett . 4, 133- 136 (1975). STOUT, R. G. and R . E. CLELAND : Effects of fusicoccin on th e activi ty of a key pH-stat enzyme, PEP-Carboxylase. - Pl ant a 139, 43--45 (1978). STOUT, R. G., K. D . JOHNSON, and D . L. RAYLE : Rap id aux in- and fusicoccin-enhanced Rb " uptake and mal at e sy nt hesis in Av ena coleo pt ile sect io ns. - Planta 139, 35-41 (1978). VANDERHOEF, L. N., J. S. FINDLEY, J. J. BURKE, and W. E. BLiZZWARD: Aux in has no eff ect on modification of ex te rn al pH by soybean hypocotyl cells. - Plant Physiol , 59, 1000-1003 (1977).
M. BOTTGER, Institut fur All gemeine Botanik der Universitar Hamburg, jungiusstrafle 6, D-2000 Hamburg 36.
Z. Pflanzenph ysiol. Rd. 99. S. 89-93. 1980.