Photochemistry and Photobiology, 1975, Vol. 22, pp. 139-144.
Pergamon Press. Printed in Great Britain
STUDIES ON HOOK-OPENING IN PHASEOLUS I/ ULGARIS L. BY SELECTIVE R/FR PRETREATMENTS OF EMBRYONIC AXIS AND PRIMARY LEAVES* R. CAUBERGS and J. A. DE GREEF Laboratory of Plant Physiology, IJniversity of Antwerpen, R.U.C.A., Groenenborgerlaan. 171, B-2020 Antwerpen, Belgium (Received 30 December 1974; accepted 18 June 1975)
Abstract-Hook-opening of etiolated bean seedlings was studied by inductive light pretreatments, selectively applied to the embryonic axis and to the primary leaves. Statistical analysis of the experimental results substantiated that hook unbending in intact plants is mutually influenced by both organs. When the leaves were excised immediately after their selective preirradiation, hook opening was the same as in the intact control series. Far red light could only fully reverse a red inductive effect when both light qualities were administered to the intact plant. When the cotyledons were removed prior to the light treatments the results did not change qualitatively. Our data indicate that a very precise, highly ordered biophysical recognition system of light signals exists in plants. The transmission of these signals between different organs is very rapid and phytochrome-mediated. INTRODUCTION
It has been known for sometime that different organs cooperate during plant development. Apical dominance and flowering induction are the best known phenomena in this respect. The initiating studies of Thimann and Skoog (1933, 1934) on bud release and of Knott (1934) on the perception of the photoperiodic stimulus have stimulated a good deal of work in these topics during the last decade. Although we are now provided with a substantial amount of experimental data on these morphogenic phenomena, little is known as to how this inter-organ dependency is regulated. In most studies of plant morphogenesis photoreactions in organs have been evocated by in situ illumination. In previous papers we could demonstrate the existence of a phytochrome-mediated inter-organ cooperation between embryonic axis and primary leaves of Phaseolus vulgaris L. in relation to leaf greening (De Greef and Caubergs, 1971, 1972. 1973). These findings confirmed our assumption that an intact plant has to be considered as an integrated system with respect to its overall developmental pattern. Therefore, we decided to search for other inter-organ dependent phenomena of plant development during the transition of heterotrophous to autotrophous growth of etiolated seedlings. Unbending of the hypocotyhr hook of etiolated bean seedlings was found to be under phytochrome control (Klein et al., 1956; Withrow et al., 1957). These authors used excised *The results presented in this paper are part of the scientific work for the doctor’s degree (doctor scientiae. dr. sc.) of R. Caubergs under Prof. J. De Greef.
hooks in their experimental procedure, thus examining the effect of phytochrome present in the hook region. With this background in mind, experiments have been designed to investigate the effect of different light pretreatments on hook opening when the light was selectively applied either to the embryonic axis or to the primary leaves. In this way we have tried to provide further evidence for the existence of a transmission system of light signals between different organs during plant development.
MATERIALS AND METHODS
Seedlings of Phaseolus uulgaris L. cv. Limburg were cultivated as described elsewhere (De Greef and Caubergs, 1972). In all experiments, 8-day old, etiolated bean plants were selected for zero degree angles. Only those plants with the hypocotyl doubled back and paralleled to the plumule were used. At this developmental stage of the seedlings, the hook region, the cotyledons. the plumule and the primary leaves are very close to each other. In order to obtain an effective shielding of the hook region and to facilitate manipulation of the material during the experiments the tops of the plants were cut off at 4-cm underneath the elbow of the hypocotyl. According to Powell and Morgan (1970), the lower hypocotyl and root tissues have little or no effect on opening of bean hooks. The seedlings were selected, cut and manipulated under a dim green safe light (Withrow and Price, 1957). Monochromatic red and far-red sources (De Greef and Caubergs, 1973) were used at an irradiance of OSOWrn-’ (659nm) and 0.57 Wm-’ (731 nm), respectively. In Fig. 1 light shielding of the hooks (Fig. 1,a) and of the primary leaves (Fig. 1,b) is illustrated. After inductive light pretreatments the organs were unshielded and the tops were placed in a darkroom, regulated for constant temperature (21°C) and
139
R. CAUBERGS and J. A. DE GREEF
140
n
hook
1
10
lhaicclip
hypocotyl
I
'black
leit
black stick tape black cardboard
I , I 1, I
I
Figure I (a). Light shielding of the hook region (embryonic axis). All parts of the seedling top, except the primary leaves, were wrapped in a husk of black stick-tape lined at the inside with soft black cardboard. The region of the husk where the primary leaves were protruding was faced with black felt to prevent damage. These felt strips were pressed together by a hair clip.
Figure 1 (b). Light shielding of the primary leaves. The primary leaves were shielded from the light by the same husk as described in Fig. la. huinidity (So"/,) so that straightening of the hook could occur freely. Tops were placed upright in beakers and soaked with their stems in tap water. In this way secondary effects on orientation of the hook tissue due to gravity was avoided (Powell and Morgan, 1970). The use of closed recipients as petri dishes was rejected to prevent accumulation of endogenously produced ethylene by which hook opening can be inhibited (Kang et a!., 1967). After 40-48 h of dark incubation the degree of hook opening was measured with a goniometer. Primary leaves and cotyledons were cut off, the isolated hooks were fixed on paper and the tangents were drawn to the plant parts forming the hook angle. Several factors influenced the variability (Sm) of hook opening between individual measurements. First, selection of bean seedlings for zero degree angle is rather arbitrary and does not guarantee the same response for each treatment. Secondly, the number of seedlings used in each experimental run was limited to 20 since the light spot obtained from the monochromatic sources was rather
* Abbreviations: R :
red light; FR: far-red light.
small. These restrictions made conclusions based upon the mean of individual results only justified for clearcut differences as in the classical experiments of Klein et al. (1956). In their study differences between the means of dark controls, R-irradiated and R/FR irradiated tops hold convincing evidence for phytochrome involvement.* In our approach we were dealing with gradual effects of hook opening and with overlapping standard errors between the means of individual experimental series. Therefore, we have repeated each experiment several times and subjected our data to the analysis of variance and to a Student-Newman-Keuls multiple range test (Woolf, 1968). The phytochrome in the hook tissue was assayed with a dual-wavelength difference photometer (Butler et al., 1963), which measured the optical density difference, AOD, between 730 and 800 nm in order to eliminate the optical density changes due to protochlorophylI(ide) transformations. ProtochlorophyIl(ide) and chlorophyll(ide) concentrations were calculated according to the formulas of Anderson and Boardman (1964).
RESULTS
In all experiments described, several combinations of selective light pretreatments on different plant parts were used to examine carefully the effect of light in the hook response. In each experiment the treatments are indicated by the following figures and symbols: @ dark control, @ R control; 5min R given to the unshielded plant top, @ R/FR control; same as @ but 5min R immediately followed by 10min FR, @ R on leaf; 5 min R selectively given to the primary leaves, while the embryonic axis (hook region) was shielded, @ R/FR on leaf; same as'@ but 5 rnin R followed by 10min FR, @ R on hook; 5 rnin R selectively applied to the hook region, while the leaves were shielded, @ R/FR on hook; same as @ but 5 rnin R followed by 10min FR, @ total R + FR on hook; 5 rnin R given to the whole top, followed by 10min FR selectively applied to the hook while the leaves were shielded, @ total R + FR on leaf; 5 rnin R given to the whole top, followed by 10min FR selectively applied to the leaves while the hook region was shielded, @ R on hook + FR on leaf; 5 rnin R selectively applied to the hook region, while the leaves are shielded, followed by 10min F R selectively given to the leaves while the hook region was shielded, @ R on leaf + FR on hook; 5 rnin R selectively applied to the leaves, while the hook 'region was shielded, followed by 1Omin FR selectively given to the hook region while the leaves were shielded.
Hook opening by selective R and RIFR pretreattnents of the primary leaves
In a first series of experiments the effect of a selective light pretreatment of the primary leaves on hook
Hook-opening and selective light pretreatments
141
Table 1. R/FR effects on hook opening by full preillumination of intact tops, selective pretreatment of primary leaves and excision of the leaves after their light treatment. For explanation of figures and symbols: see Results. Hook opening was measured 44 h after the light treatments.@ and @ are respectively the same as @ and 0. but the primary leaves were cut off immediately after their preillumination.
N"
LIGHT TREATMENTS
DIFFERENT POPULATIONS BY STAT I ST I CAL CLASS IF I CATION
DEGREE OF HOOKOPENING MEAN VALUE AND STANDARD D E V I A T I O N
1
DARK CONTROL
2
R CONTROL R/FR CONTROL
3 4 5
R ON
LEAVES
R/FR
ON LEAVES
4'
LEAVES E X C I S E D AFTER
5'
LEAVES E X C I S E D AFTER
51 142 51 100 99 112 93
R R/FR
f
22 29 22 20 16 33
t
18
t f
f f
f
I 111
I 11
I1 11 I1
Table 2. Hook opening of dark-grown tops with and without primary leaves. The primary leaves of 8-day old etiolated seedling tops were cut off and the defoliated tops were kept in complete darkness together with intact tops for 48 h. At that time the degree of hook opening was measured.
EFFECT OF LEAF EXCISION ON HOOKOPENING IN DARK-GROWN TOPS DEGREE OF HOOKOPENING MEAN VALUE AND STANDARD DEVIATION
INTACT TOPS TOPS WITHOUT LEAVES
EXP,1
EXP ,2
EXP , 3
EXP 4
67 f 18 55 f 16
88 t 15
73 f 18 58 f 18
67 f 16 67 t 18
56 2 24
I
Table 3. Hook opening by selective R and R/FR pretreatments of primary leaves and embryonic axis. Figures and symbols are explained in the text. Hook opening was measured after a dark period of 44 h following the light treatment. The results of the individual experiments were evaluated by a multiple range test and grouped in populations.
No
LIGHT TREATMENTS
DEGREE OF HOOKOPENING MEAN VALUES OF I N D I V I D U A L EXPERIMENTS
1
DARK CONTROL
2 3
R CONTROL R/FR CONTROL R ON L E A F R/FR ON LEAF R ON HOOK R/FR ON HOOK TOTAL R + FR ON HOOK TOTAL R + FR ON L E A F R ON HOOK +FR ON L E A F R ON LEAF +FR ON HOOK
4 5 6 7 8 9 10
11
ExP,1
ExD,2
ExP,~
73 130 64 25 88 127 98
70 106 68 76 80
76 142 94
125
132
73 85
111 92 119 125
96 131 130 97
115 116 90
126 130
102
ExP.4
ExP,~
78 125 76 100 99 122 86 92 127
94 129 101 107 110 154 101 99 129
125
156 90
117
TOTAL MEAN
C
EXP, 78 125 81 99
101 132 94 93 126 130
100
DIFFERENT POPULATIONS BY STAT1 ST I CAL CLASSIFICATION 1 Ill I I1 I1 111 11 11
111 111 I1
R. CAUBERGS and J. A. DE GREEF
142
lations. Dark controls (seriesO) and R/FR controls belong to the same population (I) as we (series 0) found in the experiments described in Table 1. In population I1 we find the series treated selectively with R or R/FR on the leaves (series @ and 0) and those which received R/FR on the hook (series R on the whole plant top followed by FR on the hook region (series @) and R on the leaves All the series followed by FR on the hook (series 0). of this population show a degree of unbending as if they all received an R induction on their leaves. In population 111, containing the highest values for hook opening we find the R controls (series 0) together with the series in which the hook region (= embryonic axis) was exposed to R but not to FR (series 0. @ and @). When FR is given to the leaves after an R exposure of the hook, it cannot abolish this R inductive effect for unbending (series @ and 0).All the experimental series of population I11 have statistically the same degree of hook opening as if they were all induced by a selective R preillumination of the hook. The same experiments as we described in Table 3 were repeated but this time the primary leaves were removed immediately after the light pretreatments. In these results (Table 4) the same general trend is present as we found in Table 3, although more populations are statistically evaluated. The degree of hook Hook opening by selective R and R/FR pretreatment opening was smaller for each series comparable to of priinary leaves and/or embryonic axis those of Table 3 which is in agreement with the data All 11 combinations of selective light pretreatments of Table 2. Comparing the results of both series of were used in these series of experiments; each exper- experiments (Tables 3 and 4) we notice that each iment was repeated five times. In order to statistically population of Table 3 overlaps with several populaaccount for the variability of the materiat used, all tions of Table 4; population I splits into populations experimental data were subjected to an analysis of I and 11, population I1 into populations 111 and IV. variance (two factor analysis) and to a multiple range population I11 into populations IV, V and VI. test. The results are given in Table 3. In general, it appears that hook opening is reduced On a statistical basis there are three distinct popu- when the leaves are removed after the light pretreat-
opening was tested. The results of these experiments are summarized in Table 1. After 44 h of dark incubation the R/FR reversible effect on intact plant tops is fully demonstrated (142" for the R-treated ones against 51" for both R/FR series and dark controls). This is in agreement with the findings of Klein et al. (1956) and it proves phytochrome involvement. When the primary leaves were selectively pretreated with R and R/FR and then either kept on the plants or immediately removed after (series @ and 0) their irradiation (series @ and 0). we found in all 4 series the same degree of unbending. In all 4 experimental series, hook opening is markedly larger than in the dark controls, but much less than in the R controls. Using the multiple range test, it is shown in Table 1 that dark and R/FR controls belong to the same population (I),significantly different from the R controls (population 111),while all series selectively preilluminated on their leaves are grouped in an intermediate population (11). The degree of hook opening of dark controls of comparable physiological age with excised leaves did never exceed that of intact darkgrown tops (Table 2). In fact, in most experiments hook opening was less in dark grown tops when the leaves were removed.
a),
Table 4. Same experiment as presented in Table 3, but the primary leaves were cut off immediately after the light treatment of each series. Hook opening was measured after a dark period of 48 h.
-
N'
DEGREE
LIGHT TREATMENTS
-
MEAN VALUES OF I N D I V I D U A L EXPERIMENTS
Exp
I
1
~
1
DARK CONTROL
51
2 3
R
88
4 5 6 7 8 9 10 11
-
-
OF HOOKOPENING
CONTROL
R/FR R ON
CONTROL
R/FR
ON LEAF
R ON R/FR
HOOK
65 67 81 76
ON HOOK
65
R + FR ON HOOK R + FR ON L E A F HOOK + FR ON L E A F L E A F + FR ON HOOK
81 86 94
LEAF
TOTAL TOTAL
R R
ON ON
86
tXP I 2
TOTAL MEAN
DIFFERENT POPULATIONS BY STAT1ST ICAL CLASS I F I CAT I ON
64
I
101 79 82 100 106
V
II 111 , 1v IV
IV
84 100 118 113
111 111 , I V
-
Iff , IV
75
V VI
Hook-opening and selective light pretreatnients ments, especially when the leaves were shielded. The longer the leaves remained at the plant top (due to the irradiation and shielding procedures), the more the hook response was in agreement with the results obtained in Table 3. For example, compare in both Tables 3 and 4 the results of series @ and @. and '@ and In series @. the leaves were kept in the plants for a much longer period of time than in series @ and 0. In series @ the leaves were not pretreated with light and hook opening is less than in series @ where the leaves were well exposed to light. Since there was a considerable dark period between R and FR treatments in those series where either the leaves or the hook had to be shielded after the R treatment (e.g. in series @). one could wonder if there was no R escape in this series causing the larger response. When we interpolated a comparable dark interval in the R and FR sequence given to intact tops, there was still a full FR reversal of the growth effect. Similar results have been reported by Edwards and Klein (1964). Five h after an R pretreatment, F R still gives almost complete reversal of Phaseolus hypocotyl hook opening. When the cotyledons were removed from dark-grown tops before the light treatments, the same qualitative responses were obtained. The hook response was larger, but the same graded series were found as we described in Table 3 and 4.
I43
.k'
a.
EfSiency of the shielding procedure for selective light treatments The shielding technique used to demonstrate the effect of selective light treatments on hook opening is the most crucial point in this work. As described previously, light-tight husks were used to assure a black box effect (De Greef and Caubergs, 1972). We performed several tests for the detection of eventual stray-light effects. Film strips were introduced into the hook region and the hooks were subsequently shielded. After irradiation of the leaves, the strips were checked for light penetration. No effect was observed. Exposure of the leaves to continuous white light for several days did not reveal any trace of chlorophyll(ide), present in the shielded hook tissue. Direct phytochrome measurements in hook tissues of plants, being selectively irradiated on their leaves, demonstrated the presence of PR only (Fig. 2). The same short R-irradiations given to the whole plant resulted in P R / P F R transformation and a subsequent dark decay of P F R , as it was shown by Butler et al. (1 963) and by Klein et al. (1967). The most convincing evidence that our light shielding procedure was effective was found in our studies on leaf expansion (De Greef and Caubergs. unpublished). Exposing the leaves to continuous white light and shielding the rest of the plants by the same black box effect, the leaves greened normally, but they did not expand. If, in addition, the hook was exposed
I 0
I
I
I
I
I
30
60
90
120
150
180 min
Figure 2. Kinetics of phytochrome in hook tissue of etiolated Phuseolus seedlings. PFR(0)and P,,, (0)present after an initial saturating dose of red ( 5 min R) of the whole top. P, (El) and P,,, (m) present after selective R treatment of primary leaves. daily to 5 min R, leaf expansion occurred as in white light controls. Short FR on the hook abolished this R-effect. While this fact clearly indicates that the sensitivity for PFRis quite low since the small amount of P F R established by the FR does not have a significant effect, it also shows that at least the low amounts of PFRformed by FR are not formed by light piping when the leaves were selectively illuminated by continuous white light. It proves that the black body was very effective in preventing stray-light effects.
DISCUSSION
The aim of the experiments reported here was to test our hypothesis that hook opening is influenced by light-controlled inter-organ interactions as we have demonstrated previously for greening of primary bean leaves (De Greef and Caubergs, 1972, 1973). The present data are consistent with our concept of phytochrome-mediated inter-organ cooperativity. Plant organs are not independent units with respect to their growth. In a living plant with several growth centers, development is interrelated in such a manner that the growth of one center is dependent upon the growth of the others and conversely influences the growth of other growth centers. A problem much studied in this respect is the inhibition of growth of axillary buds by the apical bud on the same shoot (see review by Phillips, 1969). Another well-documented case in this field is flower initiation where the light stimulus is perceived by the leaves. Chemical messengers appear to be formed in the leaves under particular photoperiods and are translocated through the plant to the sites of morphological responses (see review by Evans, 1974).
144
R. CA~JBERCS and J. A. DE GREEF
However, the nature, the exact production site and the quantity of the active fraction of these compound(s) are still unknown. Moreover, the primary mechanism of these interdependent developmental phenomena remains an open question. In both cases of apical dominance and of flowering induction we are dealing with long-term effects (several hours, days or weeks before a physiological effect can be observed). Our data suggest that the transmission of the signal (induced by R) from the leaves to the hook region and vice versa for hook opening is very rapid. Excision of the leaves immediately after their selective pretreatment with R did not prevent hook opening. In fact, hook unbending occurred to the same extent as if the leaves stayed at the plants all the time (Table 1). However, when the leaves were removed in dark grown tops, hook opening was generally much smaller than in intact dark controls (Table 2). Thus, hook opening can be induced by selective leaf irradiation and a rapid precise inter-organ signal transmission must take place. This kind of communication between neighbouring organs cannot be explained by growth regulators. Comparing populations I1 and 111 of Table 3 we must conclude that R-activation of the hook has a larger impact on hook opening than the R-inductive effect of the leaves. We also observed that the degree of unbending is dependent upon the presence of the leaves. As shown in Table 4,the longer the leaves stayed at the plants (due to the shielding procedure preceding a selective FR treatment after the R-preirradiation) the more the hooks were unbended. This observation can be understood by assuming that after the rapid signal transmission a second, much slower process during the dark period takes place in the leaves promoting hook unbending. It is remarkable that hook opening induced by a
selective R-treatment of the leaves cannot be reversed by a subsequent FR-irradiation selectively given either to the leaves or to the hook region (Tables 1, 3 and 4). When R was applied to the whole plant top or to the hook selectively and FR was subsequently administered in a selective manner to the hook, the hooks unbended as if the leaves were only pretreated by a selective R irradiation. The same Rpretreatment followed by a selective FR-irradiation of the leaves caused hook opening to the same extent as the R-controls. These results are in obvious contrast with the classical experiments on intact plants and with the experiments of Klein (1956) using excised hooks. In these cases R/FR reversibility was complete. According to our data FR has only an R reversing effect when both light treatments are given to the system without any part being shielded. All our findings on hook opening obtained by selective light treatments lend further support to the existence of definite interorgan interactions in intact plants. Apparently, the hook response in intact plants is mutually influenced by the different organs at the plant top when they are present. Other transmissible, phytochrome-mediated light signals were also found in electrophysiological measurements with Auena coleoptiles (Newman and Briggs, 1972) and in Sinapis seedlings for lipoxygenase synthesis in the cotyledons, controlled by phytochrome located in the hook (Oelze-Karow and Mohr, 1974). Therefore, we postulate that phytochrome directs the flow of information of perceived light signals throughout the whole plant by a transmission system (plasmalemma via plasmodesmata or microtubuli) functioning as a kind of relay system. This light signal transmission from one organ to another is so fast that only a highly ordered biophysical system can be considered as responsible for the effects observed.
REFERENCES
Anderson, J. A,, and N. K. Boardman (1964) Aust. J . Bid. Sci. 17. 93-101. Butler, W. L., H. C. Lane and H. W. Siegelman (1963) Plant Physiol. 38. 514-519. De Greef, J. A,, and R. Caubergs (1971) Arch. Intern. Physiol. Biochim. 79. 826-827. De Greef, J. A., and R. Caubergs (1972) Physiol. Plantarum 26. 157-165. De Greef, J. A,, and R. Caubergs (1973) Physiol. Plantarum 28. 71-76. Edwards, J. L., and W. H. Klein (1964) Plant Physiol. 39. 1. Evans, M. L. (1974) Ann. Rev. Plant Physiol. 25. 195-223. Kang, B. G., C. S . Yocum, S. P. Burg and P. M. Kay (1967) Science 156. 958-959. Klein, W. H., R. B. Withrow and V. B. Elstad (1956) Plant Phqaiol 31. 289-294. Klein, W. H., J. L. Edwards and W. Shropshire, Jr. (1967) Plant Physiol. 42. 264270. Knott. J. E. (1934) Proc. Am. SOC.Hort. Sci. 31. 152. Newman, I. A., and W. R. Briggs (1971) Plant Physiol. 47. 1 (Supplement). Oelze-Karow, H., and H. Mohr (1974) Photochem Photobiol. 20. 151-159. Phillips, 1. D. J. (1969) In Physiology of Plant Growth and Deoelopment (Edited by M. B. Wilkins) pp. 163-202, McGraw-Hill, London. Powell, R. D., and P. W. Morgan (1970) Plant Physiol. 45. 548-552. Thimann, K. V., and F. Skoog (1933) Proc. Nut. Acad. Sci. U.S. 19. 714-716. Thimann, K. V., and F. Skoog (1934) Proc. Rad. Bid. Soc. 114. 317-339. Withrow R. B., W. H. Klein and V. B. Elstad (1957) Plant Physiol. 32. 453-462. Woolf, C. M. (1968) Principles of Biometry Van Nostrand, London.
Pergamon Press. Printed in Great Britain
STUDIES ON HOOK-OPENING IN PHASEOLUS I/ ULGARIS L. BY SELECTIVE R/FR PRETREATMENTS OF EMBRYONIC AXIS AND PRIMARY LEAVES* R. CAUBERGS and J. A. DE GREEF Laboratory of Plant Physiology, IJniversity of Antwerpen, R.U.C.A., Groenenborgerlaan. 171, B-2020 Antwerpen, Belgium (Received 30 December 1974; accepted 18 June 1975)
Abstract-Hook-opening of etiolated bean seedlings was studied by inductive light pretreatments, selectively applied to the embryonic axis and to the primary leaves. Statistical analysis of the experimental results substantiated that hook unbending in intact plants is mutually influenced by both organs. When the leaves were excised immediately after their selective preirradiation, hook opening was the same as in the intact control series. Far red light could only fully reverse a red inductive effect when both light qualities were administered to the intact plant. When the cotyledons were removed prior to the light treatments the results did not change qualitatively. Our data indicate that a very precise, highly ordered biophysical recognition system of light signals exists in plants. The transmission of these signals between different organs is very rapid and phytochrome-mediated. INTRODUCTION
It has been known for sometime that different organs cooperate during plant development. Apical dominance and flowering induction are the best known phenomena in this respect. The initiating studies of Thimann and Skoog (1933, 1934) on bud release and of Knott (1934) on the perception of the photoperiodic stimulus have stimulated a good deal of work in these topics during the last decade. Although we are now provided with a substantial amount of experimental data on these morphogenic phenomena, little is known as to how this inter-organ dependency is regulated. In most studies of plant morphogenesis photoreactions in organs have been evocated by in situ illumination. In previous papers we could demonstrate the existence of a phytochrome-mediated inter-organ cooperation between embryonic axis and primary leaves of Phaseolus vulgaris L. in relation to leaf greening (De Greef and Caubergs, 1971, 1972. 1973). These findings confirmed our assumption that an intact plant has to be considered as an integrated system with respect to its overall developmental pattern. Therefore, we decided to search for other inter-organ dependent phenomena of plant development during the transition of heterotrophous to autotrophous growth of etiolated seedlings. Unbending of the hypocotyhr hook of etiolated bean seedlings was found to be under phytochrome control (Klein et al., 1956; Withrow et al., 1957). These authors used excised *The results presented in this paper are part of the scientific work for the doctor’s degree (doctor scientiae. dr. sc.) of R. Caubergs under Prof. J. De Greef.
hooks in their experimental procedure, thus examining the effect of phytochrome present in the hook region. With this background in mind, experiments have been designed to investigate the effect of different light pretreatments on hook opening when the light was selectively applied either to the embryonic axis or to the primary leaves. In this way we have tried to provide further evidence for the existence of a transmission system of light signals between different organs during plant development.
MATERIALS AND METHODS
Seedlings of Phaseolus uulgaris L. cv. Limburg were cultivated as described elsewhere (De Greef and Caubergs, 1972). In all experiments, 8-day old, etiolated bean plants were selected for zero degree angles. Only those plants with the hypocotyl doubled back and paralleled to the plumule were used. At this developmental stage of the seedlings, the hook region, the cotyledons. the plumule and the primary leaves are very close to each other. In order to obtain an effective shielding of the hook region and to facilitate manipulation of the material during the experiments the tops of the plants were cut off at 4-cm underneath the elbow of the hypocotyl. According to Powell and Morgan (1970), the lower hypocotyl and root tissues have little or no effect on opening of bean hooks. The seedlings were selected, cut and manipulated under a dim green safe light (Withrow and Price, 1957). Monochromatic red and far-red sources (De Greef and Caubergs, 1973) were used at an irradiance of OSOWrn-’ (659nm) and 0.57 Wm-’ (731 nm), respectively. In Fig. 1 light shielding of the hooks (Fig. 1,a) and of the primary leaves (Fig. 1,b) is illustrated. After inductive light pretreatments the organs were unshielded and the tops were placed in a darkroom, regulated for constant temperature (21°C) and
139
R. CAUBERGS and J. A. DE GREEF
140
n
hook
1
10
lhaicclip
hypocotyl
I
'black
leit
black stick tape black cardboard
I , I 1, I
I
Figure I (a). Light shielding of the hook region (embryonic axis). All parts of the seedling top, except the primary leaves, were wrapped in a husk of black stick-tape lined at the inside with soft black cardboard. The region of the husk where the primary leaves were protruding was faced with black felt to prevent damage. These felt strips were pressed together by a hair clip.
Figure 1 (b). Light shielding of the primary leaves. The primary leaves were shielded from the light by the same husk as described in Fig. la. huinidity (So"/,) so that straightening of the hook could occur freely. Tops were placed upright in beakers and soaked with their stems in tap water. In this way secondary effects on orientation of the hook tissue due to gravity was avoided (Powell and Morgan, 1970). The use of closed recipients as petri dishes was rejected to prevent accumulation of endogenously produced ethylene by which hook opening can be inhibited (Kang et a!., 1967). After 40-48 h of dark incubation the degree of hook opening was measured with a goniometer. Primary leaves and cotyledons were cut off, the isolated hooks were fixed on paper and the tangents were drawn to the plant parts forming the hook angle. Several factors influenced the variability (Sm) of hook opening between individual measurements. First, selection of bean seedlings for zero degree angle is rather arbitrary and does not guarantee the same response for each treatment. Secondly, the number of seedlings used in each experimental run was limited to 20 since the light spot obtained from the monochromatic sources was rather
* Abbreviations: R :
red light; FR: far-red light.
small. These restrictions made conclusions based upon the mean of individual results only justified for clearcut differences as in the classical experiments of Klein et al. (1956). In their study differences between the means of dark controls, R-irradiated and R/FR irradiated tops hold convincing evidence for phytochrome involvement.* In our approach we were dealing with gradual effects of hook opening and with overlapping standard errors between the means of individual experimental series. Therefore, we have repeated each experiment several times and subjected our data to the analysis of variance and to a Student-Newman-Keuls multiple range test (Woolf, 1968). The phytochrome in the hook tissue was assayed with a dual-wavelength difference photometer (Butler et al., 1963), which measured the optical density difference, AOD, between 730 and 800 nm in order to eliminate the optical density changes due to protochlorophylI(ide) transformations. ProtochlorophyIl(ide) and chlorophyll(ide) concentrations were calculated according to the formulas of Anderson and Boardman (1964).
RESULTS
In all experiments described, several combinations of selective light pretreatments on different plant parts were used to examine carefully the effect of light in the hook response. In each experiment the treatments are indicated by the following figures and symbols: @ dark control, @ R control; 5min R given to the unshielded plant top, @ R/FR control; same as @ but 5min R immediately followed by 10min FR, @ R on leaf; 5 min R selectively given to the primary leaves, while the embryonic axis (hook region) was shielded, @ R/FR on leaf; same as'@ but 5 rnin R followed by 10min FR, @ R on hook; 5 rnin R selectively applied to the hook region, while the leaves were shielded, @ R/FR on hook; same as @ but 5 rnin R followed by 10min FR, @ total R + FR on hook; 5 rnin R given to the whole top, followed by 10min FR selectively applied to the hook while the leaves were shielded, @ total R + FR on leaf; 5 rnin R given to the whole top, followed by 10min FR selectively applied to the leaves while the hook region was shielded, @ R on hook + FR on leaf; 5 rnin R selectively applied to the hook region, while the leaves are shielded, followed by 10min F R selectively given to the leaves while the hook region was shielded, @ R on leaf + FR on hook; 5 rnin R selectively applied to the leaves, while the hook 'region was shielded, followed by 1Omin FR selectively given to the hook region while the leaves were shielded.
Hook opening by selective R and RIFR pretreattnents of the primary leaves
In a first series of experiments the effect of a selective light pretreatment of the primary leaves on hook
Hook-opening and selective light pretreatments
141
Table 1. R/FR effects on hook opening by full preillumination of intact tops, selective pretreatment of primary leaves and excision of the leaves after their light treatment. For explanation of figures and symbols: see Results. Hook opening was measured 44 h after the light treatments.@ and @ are respectively the same as @ and 0. but the primary leaves were cut off immediately after their preillumination.
N"
LIGHT TREATMENTS
DIFFERENT POPULATIONS BY STAT I ST I CAL CLASS IF I CATION
DEGREE OF HOOKOPENING MEAN VALUE AND STANDARD D E V I A T I O N
1
DARK CONTROL
2
R CONTROL R/FR CONTROL
3 4 5
R ON
LEAVES
R/FR
ON LEAVES
4'
LEAVES E X C I S E D AFTER
5'
LEAVES E X C I S E D AFTER
51 142 51 100 99 112 93
R R/FR
f
22 29 22 20 16 33
t
18
t f
f f
f
I 111
I 11
I1 11 I1
Table 2. Hook opening of dark-grown tops with and without primary leaves. The primary leaves of 8-day old etiolated seedling tops were cut off and the defoliated tops were kept in complete darkness together with intact tops for 48 h. At that time the degree of hook opening was measured.
EFFECT OF LEAF EXCISION ON HOOKOPENING IN DARK-GROWN TOPS DEGREE OF HOOKOPENING MEAN VALUE AND STANDARD DEVIATION
INTACT TOPS TOPS WITHOUT LEAVES
EXP,1
EXP ,2
EXP , 3
EXP 4
67 f 18 55 f 16
88 t 15
73 f 18 58 f 18
67 f 16 67 t 18
56 2 24
I
Table 3. Hook opening by selective R and R/FR pretreatments of primary leaves and embryonic axis. Figures and symbols are explained in the text. Hook opening was measured after a dark period of 44 h following the light treatment. The results of the individual experiments were evaluated by a multiple range test and grouped in populations.
No
LIGHT TREATMENTS
DEGREE OF HOOKOPENING MEAN VALUES OF I N D I V I D U A L EXPERIMENTS
1
DARK CONTROL
2 3
R CONTROL R/FR CONTROL R ON L E A F R/FR ON LEAF R ON HOOK R/FR ON HOOK TOTAL R + FR ON HOOK TOTAL R + FR ON L E A F R ON HOOK +FR ON L E A F R ON LEAF +FR ON HOOK
4 5 6 7 8 9 10
11
ExP,1
ExD,2
ExP,~
73 130 64 25 88 127 98
70 106 68 76 80
76 142 94
125
132
73 85
111 92 119 125
96 131 130 97
115 116 90
126 130
102
ExP.4
ExP,~
78 125 76 100 99 122 86 92 127
94 129 101 107 110 154 101 99 129
125
156 90
117
TOTAL MEAN
C
EXP, 78 125 81 99
101 132 94 93 126 130
100
DIFFERENT POPULATIONS BY STAT1 ST I CAL CLASSIFICATION 1 Ill I I1 I1 111 11 11
111 111 I1
R. CAUBERGS and J. A. DE GREEF
142
lations. Dark controls (seriesO) and R/FR controls belong to the same population (I) as we (series 0) found in the experiments described in Table 1. In population I1 we find the series treated selectively with R or R/FR on the leaves (series @ and 0) and those which received R/FR on the hook (series R on the whole plant top followed by FR on the hook region (series @) and R on the leaves All the series followed by FR on the hook (series 0). of this population show a degree of unbending as if they all received an R induction on their leaves. In population 111, containing the highest values for hook opening we find the R controls (series 0) together with the series in which the hook region (= embryonic axis) was exposed to R but not to FR (series 0. @ and @). When FR is given to the leaves after an R exposure of the hook, it cannot abolish this R inductive effect for unbending (series @ and 0).All the experimental series of population I11 have statistically the same degree of hook opening as if they were all induced by a selective R preillumination of the hook. The same experiments as we described in Table 3 were repeated but this time the primary leaves were removed immediately after the light pretreatments. In these results (Table 4) the same general trend is present as we found in Table 3, although more populations are statistically evaluated. The degree of hook Hook opening by selective R and R/FR pretreatment opening was smaller for each series comparable to of priinary leaves and/or embryonic axis those of Table 3 which is in agreement with the data All 11 combinations of selective light pretreatments of Table 2. Comparing the results of both series of were used in these series of experiments; each exper- experiments (Tables 3 and 4) we notice that each iment was repeated five times. In order to statistically population of Table 3 overlaps with several populaaccount for the variability of the materiat used, all tions of Table 4; population I splits into populations experimental data were subjected to an analysis of I and 11, population I1 into populations 111 and IV. variance (two factor analysis) and to a multiple range population I11 into populations IV, V and VI. test. The results are given in Table 3. In general, it appears that hook opening is reduced On a statistical basis there are three distinct popu- when the leaves are removed after the light pretreat-
opening was tested. The results of these experiments are summarized in Table 1. After 44 h of dark incubation the R/FR reversible effect on intact plant tops is fully demonstrated (142" for the R-treated ones against 51" for both R/FR series and dark controls). This is in agreement with the findings of Klein et al. (1956) and it proves phytochrome involvement. When the primary leaves were selectively pretreated with R and R/FR and then either kept on the plants or immediately removed after (series @ and 0) their irradiation (series @ and 0). we found in all 4 series the same degree of unbending. In all 4 experimental series, hook opening is markedly larger than in the dark controls, but much less than in the R controls. Using the multiple range test, it is shown in Table 1 that dark and R/FR controls belong to the same population (I),significantly different from the R controls (population 111),while all series selectively preilluminated on their leaves are grouped in an intermediate population (11). The degree of hook opening of dark controls of comparable physiological age with excised leaves did never exceed that of intact darkgrown tops (Table 2). In fact, in most experiments hook opening was less in dark grown tops when the leaves were removed.
a),
Table 4. Same experiment as presented in Table 3, but the primary leaves were cut off immediately after the light treatment of each series. Hook opening was measured after a dark period of 48 h.
-
N'
DEGREE
LIGHT TREATMENTS
-
MEAN VALUES OF I N D I V I D U A L EXPERIMENTS
Exp
I
1
~
1
DARK CONTROL
51
2 3
R
88
4 5 6 7 8 9 10 11
-
-
OF HOOKOPENING
CONTROL
R/FR R ON
CONTROL
R/FR
ON LEAF
R ON R/FR
HOOK
65 67 81 76
ON HOOK
65
R + FR ON HOOK R + FR ON L E A F HOOK + FR ON L E A F L E A F + FR ON HOOK
81 86 94
LEAF
TOTAL TOTAL
R R
ON ON
86
tXP I 2
TOTAL MEAN
DIFFERENT POPULATIONS BY STAT1ST ICAL CLASS I F I CAT I ON
64
I
101 79 82 100 106
V
II 111 , 1v IV
IV
84 100 118 113
111 111 , I V
-
Iff , IV
75
V VI
Hook-opening and selective light pretreatnients ments, especially when the leaves were shielded. The longer the leaves remained at the plant top (due to the irradiation and shielding procedures), the more the hook response was in agreement with the results obtained in Table 3. For example, compare in both Tables 3 and 4 the results of series @ and @. and '@ and In series @. the leaves were kept in the plants for a much longer period of time than in series @ and 0. In series @ the leaves were not pretreated with light and hook opening is less than in series @ where the leaves were well exposed to light. Since there was a considerable dark period between R and FR treatments in those series where either the leaves or the hook had to be shielded after the R treatment (e.g. in series @). one could wonder if there was no R escape in this series causing the larger response. When we interpolated a comparable dark interval in the R and FR sequence given to intact tops, there was still a full FR reversal of the growth effect. Similar results have been reported by Edwards and Klein (1964). Five h after an R pretreatment, F R still gives almost complete reversal of Phaseolus hypocotyl hook opening. When the cotyledons were removed from dark-grown tops before the light treatments, the same qualitative responses were obtained. The hook response was larger, but the same graded series were found as we described in Table 3 and 4.
I43
.k'
a.
EfSiency of the shielding procedure for selective light treatments The shielding technique used to demonstrate the effect of selective light treatments on hook opening is the most crucial point in this work. As described previously, light-tight husks were used to assure a black box effect (De Greef and Caubergs, 1972). We performed several tests for the detection of eventual stray-light effects. Film strips were introduced into the hook region and the hooks were subsequently shielded. After irradiation of the leaves, the strips were checked for light penetration. No effect was observed. Exposure of the leaves to continuous white light for several days did not reveal any trace of chlorophyll(ide), present in the shielded hook tissue. Direct phytochrome measurements in hook tissues of plants, being selectively irradiated on their leaves, demonstrated the presence of PR only (Fig. 2). The same short R-irradiations given to the whole plant resulted in P R / P F R transformation and a subsequent dark decay of P F R , as it was shown by Butler et al. (1 963) and by Klein et al. (1967). The most convincing evidence that our light shielding procedure was effective was found in our studies on leaf expansion (De Greef and Caubergs. unpublished). Exposing the leaves to continuous white light and shielding the rest of the plants by the same black box effect, the leaves greened normally, but they did not expand. If, in addition, the hook was exposed
I 0
I
I
I
I
I
30
60
90
120
150
180 min
Figure 2. Kinetics of phytochrome in hook tissue of etiolated Phuseolus seedlings. PFR(0)and P,,, (0)present after an initial saturating dose of red ( 5 min R) of the whole top. P, (El) and P,,, (m) present after selective R treatment of primary leaves. daily to 5 min R, leaf expansion occurred as in white light controls. Short FR on the hook abolished this R-effect. While this fact clearly indicates that the sensitivity for PFRis quite low since the small amount of P F R established by the FR does not have a significant effect, it also shows that at least the low amounts of PFRformed by FR are not formed by light piping when the leaves were selectively illuminated by continuous white light. It proves that the black body was very effective in preventing stray-light effects.
DISCUSSION
The aim of the experiments reported here was to test our hypothesis that hook opening is influenced by light-controlled inter-organ interactions as we have demonstrated previously for greening of primary bean leaves (De Greef and Caubergs, 1972, 1973). The present data are consistent with our concept of phytochrome-mediated inter-organ cooperativity. Plant organs are not independent units with respect to their growth. In a living plant with several growth centers, development is interrelated in such a manner that the growth of one center is dependent upon the growth of the others and conversely influences the growth of other growth centers. A problem much studied in this respect is the inhibition of growth of axillary buds by the apical bud on the same shoot (see review by Phillips, 1969). Another well-documented case in this field is flower initiation where the light stimulus is perceived by the leaves. Chemical messengers appear to be formed in the leaves under particular photoperiods and are translocated through the plant to the sites of morphological responses (see review by Evans, 1974).
144
R. CA~JBERCS and J. A. DE GREEF
However, the nature, the exact production site and the quantity of the active fraction of these compound(s) are still unknown. Moreover, the primary mechanism of these interdependent developmental phenomena remains an open question. In both cases of apical dominance and of flowering induction we are dealing with long-term effects (several hours, days or weeks before a physiological effect can be observed). Our data suggest that the transmission of the signal (induced by R) from the leaves to the hook region and vice versa for hook opening is very rapid. Excision of the leaves immediately after their selective pretreatment with R did not prevent hook opening. In fact, hook unbending occurred to the same extent as if the leaves stayed at the plants all the time (Table 1). However, when the leaves were removed in dark grown tops, hook opening was generally much smaller than in intact dark controls (Table 2). Thus, hook opening can be induced by selective leaf irradiation and a rapid precise inter-organ signal transmission must take place. This kind of communication between neighbouring organs cannot be explained by growth regulators. Comparing populations I1 and 111 of Table 3 we must conclude that R-activation of the hook has a larger impact on hook opening than the R-inductive effect of the leaves. We also observed that the degree of unbending is dependent upon the presence of the leaves. As shown in Table 4,the longer the leaves stayed at the plants (due to the shielding procedure preceding a selective FR treatment after the R-preirradiation) the more the hooks were unbended. This observation can be understood by assuming that after the rapid signal transmission a second, much slower process during the dark period takes place in the leaves promoting hook unbending. It is remarkable that hook opening induced by a
selective R-treatment of the leaves cannot be reversed by a subsequent FR-irradiation selectively given either to the leaves or to the hook region (Tables 1, 3 and 4). When R was applied to the whole plant top or to the hook selectively and FR was subsequently administered in a selective manner to the hook, the hooks unbended as if the leaves were only pretreated by a selective R irradiation. The same Rpretreatment followed by a selective FR-irradiation of the leaves caused hook opening to the same extent as the R-controls. These results are in obvious contrast with the classical experiments on intact plants and with the experiments of Klein (1956) using excised hooks. In these cases R/FR reversibility was complete. According to our data FR has only an R reversing effect when both light treatments are given to the system without any part being shielded. All our findings on hook opening obtained by selective light treatments lend further support to the existence of definite interorgan interactions in intact plants. Apparently, the hook response in intact plants is mutually influenced by the different organs at the plant top when they are present. Other transmissible, phytochrome-mediated light signals were also found in electrophysiological measurements with Auena coleoptiles (Newman and Briggs, 1972) and in Sinapis seedlings for lipoxygenase synthesis in the cotyledons, controlled by phytochrome located in the hook (Oelze-Karow and Mohr, 1974). Therefore, we postulate that phytochrome directs the flow of information of perceived light signals throughout the whole plant by a transmission system (plasmalemma via plasmodesmata or microtubuli) functioning as a kind of relay system. This light signal transmission from one organ to another is so fast that only a highly ordered biophysical system can be considered as responsible for the effects observed.
REFERENCES
Anderson, J. A,, and N. K. Boardman (1964) Aust. J . Bid. Sci. 17. 93-101. Butler, W. L., H. C. Lane and H. W. Siegelman (1963) Plant Physiol. 38. 514-519. De Greef, J. A,, and R. Caubergs (1971) Arch. Intern. Physiol. Biochim. 79. 826-827. De Greef, J. A., and R. Caubergs (1972) Physiol. Plantarum 26. 157-165. De Greef, J. A,, and R. Caubergs (1973) Physiol. Plantarum 28. 71-76. Edwards, J. L., and W. H. Klein (1964) Plant Physiol. 39. 1. Evans, M. L. (1974) Ann. Rev. Plant Physiol. 25. 195-223. Kang, B. G., C. S . Yocum, S. P. Burg and P. M. Kay (1967) Science 156. 958-959. Klein, W. H., R. B. Withrow and V. B. Elstad (1956) Plant Phqaiol 31. 289-294. Klein, W. H., J. L. Edwards and W. Shropshire, Jr. (1967) Plant Physiol. 42. 264270. Knott. J. E. (1934) Proc. Am. SOC.Hort. Sci. 31. 152. Newman, I. A., and W. R. Briggs (1971) Plant Physiol. 47. 1 (Supplement). Oelze-Karow, H., and H. Mohr (1974) Photochem Photobiol. 20. 151-159. Phillips, 1. D. J. (1969) In Physiology of Plant Growth and Deoelopment (Edited by M. B. Wilkins) pp. 163-202, McGraw-Hill, London. Powell, R. D., and P. W. Morgan (1970) Plant Physiol. 45. 548-552. Thimann, K. V., and F. Skoog (1933) Proc. Nut. Acad. Sci. U.S. 19. 714-716. Thimann, K. V., and F. Skoog (1934) Proc. Rad. Bid. Soc. 114. 317-339. Withrow R. B., W. H. Klein and V. B. Elstad (1957) Plant Physiol. 32. 453-462. Woolf, C. M. (1968) Principles of Biometry Van Nostrand, London.
