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Effect of Indoleacetic Acid on Wound-activated Ribonuclease in Potato Tuber Slices
Effect of Indoleacetic Acid on Wound-activated Ribonuclease in Potato Tuber Slices MARlA CLARA ISOLA and LUIS FRANZONI Departamento de Biologfa Molecular, Facultad de Ciencias Exactas, Flsico-Quimicas y Naturales, Universidad Nacional de Rio Cuarto, 5600 Rio Cuarto, Argentina Received October 15, 1982 . Accepted January 10, 1983
Summary Aging causes a large increase in ribonuclease (RNase) activity in slices from growing, mature or sprouting potato tubers. The presence of 10- 5 M indole-3-acetic acid (IAA) in the aging medium partially inhibits the activation of RNase if applied to tissue sections from growing potato tubers, but increases it in discs of mature or sprouting tubers. In all physiological stages analyzed, the aging process modifies the electrophoretic pattern of RNase in the same way, with a considerable increase in only one of the bands of activity. Incubation of tissues from growing tubers in IAA resulted in a rise in the same band as in similar discs incubated in water. On the other hand, in discs of mature or sprouting tubers, aging in IAA produces a general increase in the electrophoretic profile of RNases with respect to the controls. These results are consistent with the view that, according to the physiological stage of the tubers, the different RNase isozymes exhibit a different sensivity to auxin. Alternatively, a dose-dependent effect of the hormone is not ruled out. Whereas exogenously applied IAA increases the activation of RNase in discs of mature tubers at concentrations up to 10-4 M, higher concentrations are inhibitory. However, since the comparative experiments were performed in a fixed concentration of IAA (10- 5 M), the possible influence of endogenous auxin levels is discussed.
Key words: Solanum tuberosum, ageing, auxin, ribonuclease.
Introduction Aging of tissue slices from plant storage organs in a moist atmosphere induces a number of metabolic changes within the tissue. The question of the molecular mechanism by which these changes are brought about after slicing remains unsolved, but the possible involvement of plant growth regulators in the wound responses has been repeatedly discussed (Kahl, 1973; Imaseki et aI., 1968; Van Steveninck, 1975). Actually, gibberellic acid (GA3) (Rappaport and Sachs, 1967), ethylene (Okazawa, 1974) and IAA (Conrad and Kahn, 1975; Hemberg, 1947) are rapidly synthesized or activated after slicing potato tuber tissue. Although no experimental support is presently at hand concerning wound-synthesized phytohormones as aging effectors, there is no doubt that exogenous application of, e.g., gibberellin or ethylene exerts a stimulative effect on some systems (for a review see Rosenstock and Kahl, 1978). However, the response may depend on the Z. Pjlanzenphysiol. Bd. 110. S. 61-67. 1983.
62
MARiA CLARA ISOLA and LUIS FRANZONI
history and the competence of the tissue. Wielgat et aI. (1979) have reported that application of GA3 to potato tuber slices enhances RNA polymerase activity and template availability of chromatin over the control slices, aged whithout hormone. However, the effect of GA3 is strictly dependent on the tuber age. Slices from growing tubers are not responsive toward the hormone. The tissue becomes competent for GA3 during dormancy and sprouting. Breakdown of RNA molecules is regulated in potato tuber slices by wound enhanced activity of RNases (Pitt and Galpin, 1971; Sacher et aI., 1972). We have demonstrated, by means of gel electrophoresis, that the increased activity is largely due to one among five isozymes (Isola and Franzoni, 1981). In this investigation we have tested the effects of IAA on RNase activation, since auxin, according to several reports, has an important influence on the RNase activity in different plant tissues (for a review see Jacobsen, 1977). The present experiments aimed at finding out whether a) exogenous IAA influence the RNase activation in the potato slice system, b) IAA may act preferentially on some particular RNase isozymes and c) the effect of IAA changes with the physiological age of tubers.
Material and Methods Plant Material: Potato tubers (Solanum tuberosum L. var. Spunta) were planted in a greenhouse in April. Tubers at three physiological stages were used. Young tubers, showing rapid growth (<
Results and Discussion Aging of potato tuber slices for 16 hours (the time for maximum activation, see below), causes an important increase in RNase activity regardless of the stage of development of the tubers (Table 1). On the other hand, the effect of the presence of
z. Pjlanzenphysiol. Ed. 110. S. 61-67. 1983.
Indoleacetic acid and RNase in potato tuber
63
Table 1: Effect of aging and IAA on RNase activity in discs cut from growing and mature potato tubers. RNase activity (units/mgprotein)C) Slices aged for 16 h in: Slices from tubers
number of experiments')
tuber weight b)
Growing Mature
7 8
7.5 (0.6-19.4) 49.2 (34.1-70.5)
fresh slices
waterd)
10- 5 M IAAd)
0.21±0.02 0.18±0.02
0.80±0.02 0.59±0.02
0.56±0.04 0.71±0.01
(g)
.) 2-3 tubers were used for each experiment. b) Average weight and range (in parenthesis). Values are means + standard error. ) Data within each column are significantly different at the 0.01 level.
2
IAA during incubation of the slices depends on the physiological age of the tubers. IAA diminishes the activation of RNase in discs of growing tubers, but increases it in slices of mature tubers. At the end of the tuberization period, the inhibitory effect of IAA decreases considerably, and the RNase activity in hormonetreated discs varies below and above the control levels, until reaching a balance in the typical values of mature tubers. The higher standard error of this experimental group reflect this oscillation. The time course of RNase activity during aging is shown in Fig. 1. Application of 10- 5 M IAA to aging discs from growing tubers, reduces significantly the activity of wound-induced RNase after 10 hours of incubation (Fig. 1 A). In slices of mature tub-
c
B
0.9 A
Q/?"\
]
0.6
e Co
Cl
E
'" 0.3 ~ c
I
I~
~~!~
I
'\
.2
Q)
rn m Z
1Q~£
J
r
r\
~~~')
?'.!
a:
o
10
20
0
10
20 0 10 20 time after slicing(h)
Fig. 1: Time course of the development of RNase activity during aging of slices from: growing (A), mature (B) and sprouted (C) potato tubers, in absence (0--0) or presence (e--e) of 10-5 M IAA. Mean values of three experiments and SE.
Z. Pjlanzenphysiol. Bd. 110. S. 61-67. 1983.
64
MARiA CLARA ISOLA and LUIS FRANZONI
0
1
0
Rm
0
15 Oh
-
(A)
IAA
Hi>
QI
.~
~ 10 QI
~ ~
·c
2
QI III
cu
z
5
a:
~ 10
10
20
10
20
20
slice number Rm
0
1
0
0
15 H2O
Oh
-
( S)
IAA
QI
~ 10 III
'i .Ji!!
2 .3
·c QI
III cu 5
z
a:
~ 1
10
20
10
20
10
20
sl ice number
Fig. 2: Electrophoretic patterns of RNases of discs from growing (A) or sprouted (B) potato tubers. Oh: fresh discs; H 20: discs aged in water for 16 h; lAA: disc aged in 10- 5 M lAA for 16 h; Rm: relative mobility with respect to front marker.
ers, RNase activation is reduced in the presence of IAA during the first hours of incubation, but thereafter, about 12 to 20 hours after cutting, exceeds the values of control discs aged in water (Fig. 1 b). A similar pattern of activation is seen in slices of Z. Pflanzenphysiol. Bd. 110. S. 61-67. 1983.
Indoleacetic acid and RNase in potato tuber
65
sprouting tubers, although the stimulatory effect of IAA is quantitatively higher (Fig. 1 c). Conceivably, IAA may regulate the total RNase activity, acting on one or more RNases of the tissue. The existence of several types of RNA hydrolyzing enzymes in potato tubers has been repeatedly pointed out (Bjork, 1965; Pitt and Galpin, 1971). It may be suggested, therefore, that different effects on different RNases could explain the dual response to IAA according to the physiological age of the tubers. As has been shown previously (Isola and Franzoni, 1981), during aging of discs of mature potato tubers, the electrophoretic pattern of RNases shows a considerable increase in only one of the bands of activity, with no or very little modification in the remaining bands. The electrophoretic patterns in Figs. 2 A and 2 B show that in slices from growing or sprouting tubers, aging in water produces the activation of the isozyme with Rm around 0.75. On the other hand, discs from tubers in different developmental stages respond differently to IAA. If tissue from actively growing tubers is incubated in 10- 5 M IAA, the activation of RNase isozyme with Rm around 0.75 is reduced with respect to the control aged in water, with little differences in other bands (Fig. 2 A). In contrast, the same band reaches its maximum activity in discs aged in IAA when theses proceeds from sprouting tubers, although in this case, a general increase of the whole electrophoretic profile is observed (Fig. 2 B). The response of discs from mature tubers to IAA, with respect to the electrophoretic patterns of their RNases, is quite similar to that of sprouting tubers (not shown). The above results support, at least partially, the hypothesis that the various nucleases of potato tubers may have different sensitivities to auxin in different stages of tuber development. An alternative explanation of the age-dependent effect of IAA could be based on the already known fact that the effect of the auxin on the RNase activity may vary according to the concentration of the auxin applied (Shanon et aI., 1964).
C 0.9
.... Q)
e Q.
01
.E
0.6 control without IAA
.~ c:
:::I
Q)
'"
~03 £r
Fig. 3: Effect of IAA in the medium on the development of RNase activity in potato tuber discs during aging. Mean values of three experiments and SE.
7
6
5
4 3 2 -log IAA (M)
z. Pjlanzenphysiol. Bd. 110. S. 61-67. 1983.
66
MARiA CLARA ISOLA and LUIS FRANZONI
In our experimental system, in which a fixed concentration of hormone is added exogenously, it is possible that the endogenous concentration of auxin could determine the tissue response. In this respect, Obata-Sasamoto and Suzuki (1979) have shown that auxin concentration in potato tubers is maximum (in the order of 10- 7 mol IAA per kg, on a fresh weight basis) at the beginning of tuberization, rapidly decreasing afterwards. Therefore, when aging takes place in the presence of 10-5 M IAA, a partial blockade of RNase activation coincides with a relatively high concentration of endogenous auxin, while an increased stimulation is observed when the auxin in the tissue reaches a low level. On this basis, it may be expected that a sufficiently high dose of IAA in the aging medium would result in a decrease of RNase activation in discs of mature tubers. As can be see in Fig. 3, slices from mature tubers show an increase in the RNase activation when they are aged for 16 hours in the presence of 10-6 up to 10-4 M IAA. Only a very high concentration (10- 2 M) of auxin is significantly inhibitory. Interpretation of this experiment, however, leaves some uncertainty, due to lack of information concerning the possible influence of certain factors such as the rate of uptake and destruction and! or conjugation of exogenous IAA within the tissue. Finally, it is to be noted that, in some cases, the effect of IAA can be accounted for by auxin-stimulated ethylene production (see review in Abeles, 1973). Many enzymes have seen found to be either increased or reduced in activity following the application of ethylene to tissue sections (see e.g. Sacher and Engstrom, 1977). Goto and Esashi (1974) found that IAA inhibited growth in zones corresponding to the hook region of Phaseolus hypocotyls, but enhanced growth in older portions of the same tissue. They concluded that the inhibition was due to an IAA-induced rise in endogenous ethylene. Such a mechanism cannot be ruled out in our system, and further investigation is needed to test this possibility. Acknowledgements We wish to express our sincere thanks for valuable suggestions to Prof. Dr. R. Tizio. We are also indebted to Mr. E. Franzone for technical assistance and to Mrs. G. Bauducco who translated this work. Supported by grants of the Subsecretarla de Ciencia y Tecnica, Consejo de Investigaciones de la Provincia de C6rdoba and Consejo Nacional de Investigaciones Cientfficas y Tecnicas.
References ABELES, F. B.: Ethylene in Plant Biology. Academic Press, New York, 1973. BJORK, N .: Purification of two endonucleases from potato tubers. Biochim. Biophys. Acta 95, 652-662 (1965). CONRAD, K. und B. KOHN: Zunahme von Cytokinin and Auxin in verwundetem Speichergewebe von Solanum tuherosum. Phytochem. 14, 325-328 (1975). DAVIS, B. J.: Disc electrophoresis. II. Methods and application to human serum proteins. Ann. N . Y. Acad. Sci. 121,404-427 (1964).
Z. Pjlanzenphysiol. Bd. 110. S. 61-67. 1983.
Indoleacetic acid and RNase in potato tuber
67
GOTO, N. and Y. ESASHI: Differential hormone responses in different growing zones of the bean hypocotyl. Planta 116, 225-241 (1974). IMASEKI, H., M. UCHIYAMA, and 1. UR/TANI: Effect of ethylene on the inductive increase in metabolic activities in sliced sweet potato roots. Agr. BioI. Chern. 32, 387-389 (1968). ISOLA, M. C. and L. FRANZONI: Changes in electrophoretic pattern of ribonucleases during aging of potato tuber slices. Z. Pflanzenphysiol. 103, 277-283 (1981). JACOBSEN, J. V.: Regulation of ribonucleic acid metabolism by plant hormones. Ann. Rev. Plant Physiol. 28, 537-564 (1977). KAHL, G.: Genetic and metabolic regulation in differentiating plant storage tissue cells. Bot. Rev. 39,274-299 (1973). KOCH, A. L. and S. L. PUTNAM: Sensitive biuret method for determination of protein in an impure system such as whole bacteria. Anal. Biochem. 44, 239-245 (1971). OBATA-SASAMOTO, H. and H. SUZUKI: Activities of enzymes relating to starch synthesis and endogenous levels of growth regulators in potato stolon tips during tuberization. Physiol. Plant. 45, 320-324 (1979). OKAZAWA, Y.: A relation between ethylene evolution and sprouting of potato tuber. J. Facul. Agric. Hokkaido Univ. 57, 442-454 (1974). PITT, D. and M. GALPIN: Increase in ribonuclease activity following mechanical damage to leaf and tuber tissues of Solanum tuberosum L. Plant a 101, 317-332 (1971). RApPAPORT, L. and M. SACHS: Wound-induced gibberellins. Nature 214, 1149-1150 (1967). ROSENSTOCK, G. and G. KAHL: Phytohormones and the regulation of cellular processes in aging storage tissues. In: Biochemistry of wounded plant tissues, KAHL, G. (ed.), pp. 623-67l. Walter de Gruyter, Berlin, 1978. SACHER,J. A., G. H. N. TOWERS, and D. D. DAVIES: Effect of light and aging on enzymes, particularly phenylalanine ammonia lyase, in discs of storage tissue. Phytochem. 11, 2383-2391 (1972). SACHER, J. A. and D. ENGSTROM: Ethylene regulation of wound-induced ribonuclease in turnip. (abstr). Plant Physiol. 59, sup. 38 (1977). SHANON, J. c., J. B. HANSON, and C. M. WILSON: Ribonuclease levels in the mesocotyl tissue of Zea mays as a function of 2-4 dichlorophenoxyacetic acid application. Plant Physiol. 39, 804-809 (1964). VAN STEVENINCK, R. F. M.: The washing or aging phenomenon in plant tissues. Ann. Rev. Plant Physiol. 26, 237-258 (1975). WIELGAT, B., M. WECHSELBERGER, and G. KAHL: Age-dependent variations in transcriptional response to wounding and gibberellic acid in a higher plant. Planta 147, 205-209 (1979). WILSON, C. M.: Plant Nucleases III. Polyacrylamide gel electrophoresis of corn isozymes. Plant Physiol. 48, 64-78 (1971). - Plant nucleases. Ann. Rev. Plant Physiol. 26, 187-208 (1975).
Z. Pjlanzenphysiol. Ed. 110. S. 61-67. 1983.
Summary Aging causes a large increase in ribonuclease (RNase) activity in slices from growing, mature or sprouting potato tubers. The presence of 10- 5 M indole-3-acetic acid (IAA) in the aging medium partially inhibits the activation of RNase if applied to tissue sections from growing potato tubers, but increases it in discs of mature or sprouting tubers. In all physiological stages analyzed, the aging process modifies the electrophoretic pattern of RNase in the same way, with a considerable increase in only one of the bands of activity. Incubation of tissues from growing tubers in IAA resulted in a rise in the same band as in similar discs incubated in water. On the other hand, in discs of mature or sprouting tubers, aging in IAA produces a general increase in the electrophoretic profile of RNases with respect to the controls. These results are consistent with the view that, according to the physiological stage of the tubers, the different RNase isozymes exhibit a different sensivity to auxin. Alternatively, a dose-dependent effect of the hormone is not ruled out. Whereas exogenously applied IAA increases the activation of RNase in discs of mature tubers at concentrations up to 10-4 M, higher concentrations are inhibitory. However, since the comparative experiments were performed in a fixed concentration of IAA (10- 5 M), the possible influence of endogenous auxin levels is discussed.
Key words: Solanum tuberosum, ageing, auxin, ribonuclease.
Introduction Aging of tissue slices from plant storage organs in a moist atmosphere induces a number of metabolic changes within the tissue. The question of the molecular mechanism by which these changes are brought about after slicing remains unsolved, but the possible involvement of plant growth regulators in the wound responses has been repeatedly discussed (Kahl, 1973; Imaseki et aI., 1968; Van Steveninck, 1975). Actually, gibberellic acid (GA3) (Rappaport and Sachs, 1967), ethylene (Okazawa, 1974) and IAA (Conrad and Kahn, 1975; Hemberg, 1947) are rapidly synthesized or activated after slicing potato tuber tissue. Although no experimental support is presently at hand concerning wound-synthesized phytohormones as aging effectors, there is no doubt that exogenous application of, e.g., gibberellin or ethylene exerts a stimulative effect on some systems (for a review see Rosenstock and Kahl, 1978). However, the response may depend on the Z. Pjlanzenphysiol. Bd. 110. S. 61-67. 1983.
62
MARiA CLARA ISOLA and LUIS FRANZONI
history and the competence of the tissue. Wielgat et aI. (1979) have reported that application of GA3 to potato tuber slices enhances RNA polymerase activity and template availability of chromatin over the control slices, aged whithout hormone. However, the effect of GA3 is strictly dependent on the tuber age. Slices from growing tubers are not responsive toward the hormone. The tissue becomes competent for GA3 during dormancy and sprouting. Breakdown of RNA molecules is regulated in potato tuber slices by wound enhanced activity of RNases (Pitt and Galpin, 1971; Sacher et aI., 1972). We have demonstrated, by means of gel electrophoresis, that the increased activity is largely due to one among five isozymes (Isola and Franzoni, 1981). In this investigation we have tested the effects of IAA on RNase activation, since auxin, according to several reports, has an important influence on the RNase activity in different plant tissues (for a review see Jacobsen, 1977). The present experiments aimed at finding out whether a) exogenous IAA influence the RNase activation in the potato slice system, b) IAA may act preferentially on some particular RNase isozymes and c) the effect of IAA changes with the physiological age of tubers.
Material and Methods Plant Material: Potato tubers (Solanum tuberosum L. var. Spunta) were planted in a greenhouse in April. Tubers at three physiological stages were used. Young tubers, showing rapid growth (<
Results and Discussion Aging of potato tuber slices for 16 hours (the time for maximum activation, see below), causes an important increase in RNase activity regardless of the stage of development of the tubers (Table 1). On the other hand, the effect of the presence of
z. Pjlanzenphysiol. Ed. 110. S. 61-67. 1983.
Indoleacetic acid and RNase in potato tuber
63
Table 1: Effect of aging and IAA on RNase activity in discs cut from growing and mature potato tubers. RNase activity (units/mgprotein)C) Slices aged for 16 h in: Slices from tubers
number of experiments')
tuber weight b)
Growing Mature
7 8
7.5 (0.6-19.4) 49.2 (34.1-70.5)
fresh slices
waterd)
10- 5 M IAAd)
0.21±0.02 0.18±0.02
0.80±0.02 0.59±0.02
0.56±0.04 0.71±0.01
(g)
.) 2-3 tubers were used for each experiment. b) Average weight and range (in parenthesis). Values are means + standard error. ) Data within each column are significantly different at the 0.01 level.
2
IAA during incubation of the slices depends on the physiological age of the tubers. IAA diminishes the activation of RNase in discs of growing tubers, but increases it in slices of mature tubers. At the end of the tuberization period, the inhibitory effect of IAA decreases considerably, and the RNase activity in hormonetreated discs varies below and above the control levels, until reaching a balance in the typical values of mature tubers. The higher standard error of this experimental group reflect this oscillation. The time course of RNase activity during aging is shown in Fig. 1. Application of 10- 5 M IAA to aging discs from growing tubers, reduces significantly the activity of wound-induced RNase after 10 hours of incubation (Fig. 1 A). In slices of mature tub-
c
B
0.9 A
Q/?"\
]
0.6
e Co
Cl
E
'" 0.3 ~ c
I
I~
~~!~
I
'\
.2
Q)
rn m Z
1Q~£
J
r
r\
~~~')
?'.!
a:
o
10
20
0
10
20 0 10 20 time after slicing(h)
Fig. 1: Time course of the development of RNase activity during aging of slices from: growing (A), mature (B) and sprouted (C) potato tubers, in absence (0--0) or presence (e--e) of 10-5 M IAA. Mean values of three experiments and SE.
Z. Pjlanzenphysiol. Bd. 110. S. 61-67. 1983.
64
MARiA CLARA ISOLA and LUIS FRANZONI
0
1
0
Rm
0
15 Oh
-
(A)
IAA
Hi>
QI
.~
~ 10 QI
~ ~
·c
2
QI III
cu
z
5
a:
~ 10
10
20
10
20
20
slice number Rm
0
1
0
0
15 H2O
Oh
-
( S)
IAA
QI
~ 10 III
'i .Ji!!
2 .3
·c QI
III cu 5
z
a:
~ 1
10
20
10
20
10
20
sl ice number
Fig. 2: Electrophoretic patterns of RNases of discs from growing (A) or sprouted (B) potato tubers. Oh: fresh discs; H 20: discs aged in water for 16 h; lAA: disc aged in 10- 5 M lAA for 16 h; Rm: relative mobility with respect to front marker.
ers, RNase activation is reduced in the presence of IAA during the first hours of incubation, but thereafter, about 12 to 20 hours after cutting, exceeds the values of control discs aged in water (Fig. 1 b). A similar pattern of activation is seen in slices of Z. Pflanzenphysiol. Bd. 110. S. 61-67. 1983.
Indoleacetic acid and RNase in potato tuber
65
sprouting tubers, although the stimulatory effect of IAA is quantitatively higher (Fig. 1 c). Conceivably, IAA may regulate the total RNase activity, acting on one or more RNases of the tissue. The existence of several types of RNA hydrolyzing enzymes in potato tubers has been repeatedly pointed out (Bjork, 1965; Pitt and Galpin, 1971). It may be suggested, therefore, that different effects on different RNases could explain the dual response to IAA according to the physiological age of the tubers. As has been shown previously (Isola and Franzoni, 1981), during aging of discs of mature potato tubers, the electrophoretic pattern of RNases shows a considerable increase in only one of the bands of activity, with no or very little modification in the remaining bands. The electrophoretic patterns in Figs. 2 A and 2 B show that in slices from growing or sprouting tubers, aging in water produces the activation of the isozyme with Rm around 0.75. On the other hand, discs from tubers in different developmental stages respond differently to IAA. If tissue from actively growing tubers is incubated in 10- 5 M IAA, the activation of RNase isozyme with Rm around 0.75 is reduced with respect to the control aged in water, with little differences in other bands (Fig. 2 A). In contrast, the same band reaches its maximum activity in discs aged in IAA when theses proceeds from sprouting tubers, although in this case, a general increase of the whole electrophoretic profile is observed (Fig. 2 B). The response of discs from mature tubers to IAA, with respect to the electrophoretic patterns of their RNases, is quite similar to that of sprouting tubers (not shown). The above results support, at least partially, the hypothesis that the various nucleases of potato tubers may have different sensitivities to auxin in different stages of tuber development. An alternative explanation of the age-dependent effect of IAA could be based on the already known fact that the effect of the auxin on the RNase activity may vary according to the concentration of the auxin applied (Shanon et aI., 1964).
C 0.9
.... Q)
e Q.
01
.E
0.6 control without IAA
.~ c:
:::I
Q)
'"
~03 £r
Fig. 3: Effect of IAA in the medium on the development of RNase activity in potato tuber discs during aging. Mean values of three experiments and SE.
7
6
5
4 3 2 -log IAA (M)
z. Pjlanzenphysiol. Bd. 110. S. 61-67. 1983.
66
MARiA CLARA ISOLA and LUIS FRANZONI
In our experimental system, in which a fixed concentration of hormone is added exogenously, it is possible that the endogenous concentration of auxin could determine the tissue response. In this respect, Obata-Sasamoto and Suzuki (1979) have shown that auxin concentration in potato tubers is maximum (in the order of 10- 7 mol IAA per kg, on a fresh weight basis) at the beginning of tuberization, rapidly decreasing afterwards. Therefore, when aging takes place in the presence of 10-5 M IAA, a partial blockade of RNase activation coincides with a relatively high concentration of endogenous auxin, while an increased stimulation is observed when the auxin in the tissue reaches a low level. On this basis, it may be expected that a sufficiently high dose of IAA in the aging medium would result in a decrease of RNase activation in discs of mature tubers. As can be see in Fig. 3, slices from mature tubers show an increase in the RNase activation when they are aged for 16 hours in the presence of 10-6 up to 10-4 M IAA. Only a very high concentration (10- 2 M) of auxin is significantly inhibitory. Interpretation of this experiment, however, leaves some uncertainty, due to lack of information concerning the possible influence of certain factors such as the rate of uptake and destruction and! or conjugation of exogenous IAA within the tissue. Finally, it is to be noted that, in some cases, the effect of IAA can be accounted for by auxin-stimulated ethylene production (see review in Abeles, 1973). Many enzymes have seen found to be either increased or reduced in activity following the application of ethylene to tissue sections (see e.g. Sacher and Engstrom, 1977). Goto and Esashi (1974) found that IAA inhibited growth in zones corresponding to the hook region of Phaseolus hypocotyls, but enhanced growth in older portions of the same tissue. They concluded that the inhibition was due to an IAA-induced rise in endogenous ethylene. Such a mechanism cannot be ruled out in our system, and further investigation is needed to test this possibility. Acknowledgements We wish to express our sincere thanks for valuable suggestions to Prof. Dr. R. Tizio. We are also indebted to Mr. E. Franzone for technical assistance and to Mrs. G. Bauducco who translated this work. Supported by grants of the Subsecretarla de Ciencia y Tecnica, Consejo de Investigaciones de la Provincia de C6rdoba and Consejo Nacional de Investigaciones Cientfficas y Tecnicas.
References ABELES, F. B.: Ethylene in Plant Biology. Academic Press, New York, 1973. BJORK, N .: Purification of two endonucleases from potato tubers. Biochim. Biophys. Acta 95, 652-662 (1965). CONRAD, K. und B. KOHN: Zunahme von Cytokinin and Auxin in verwundetem Speichergewebe von Solanum tuherosum. Phytochem. 14, 325-328 (1975). DAVIS, B. J.: Disc electrophoresis. II. Methods and application to human serum proteins. Ann. N . Y. Acad. Sci. 121,404-427 (1964).
Z. Pjlanzenphysiol. Bd. 110. S. 61-67. 1983.
Indoleacetic acid and RNase in potato tuber
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