Planta (Berl.) 90, 286--294 (1970)
Phytochrome and Hormonal Control of Expansion and Greening of Etiolated Wheat Leaves Lro~A~n B r r w ~ s , B. L o w Y s , J. A. P~,~_~so~ and P. F. WAi~I~G Botany Department, University College of Wales, Aberystwyth Received Sepember 19/November 10, 1969
Summary. UIrrolling of etiolated wheat leaf segments is stimulated by short periods of exposure to red light. Both gibberellic acid and kinetin will stimulate unrolling in the dark, whereas abscisie acid (ABA) inhibits the unrolling response to these two hormones and to red light. Exposure to 5 minutes of red light leads to a rapid increase in endogenous gibberellin levels in etiolated wheat leaves, and this increase is followed by a rapid decline. Pre-treatment with ABA inhibits the increase in gibberellin levels in response to red light, but the inhibitory effect of ABA on unrolling cannot be ascribed only to its effect on gibberellin levels. Pre-treatment with red light reduces the lag-phase in chlorophyll development when wheat leaf segments are subsequently exposed to white light; the effect of red light may be replaced by pre-treatment with kinetin, but gibberellic acid is relatively ineffective in this respect. Introduction I t has long been known t h a t leaf expansion of etiolated leaves is promoted b y light, and Parker et al. (1949) showed t h a t red light was the most effective spectral region stimulating leaf growth. I t was later demonstrated t h a t the effect of red light can be reversed by far-red light (Downs, 1955; Liverman et al., 1955), thus indicating that the phytochrome system is involved in normal leaf expansion. I n studies on wheat leaves, Virgin (1962) demonstrated that both the greening process and the leaf unrolling characteristic of graminaceous species is stimulated b y red light and reversed b y far-red light. Klein et al. (1963) have similarly demonstrated an involvement of the phytochrome system in controlling the unrolling of leaves of maize. Attempts have been made to relate these radiation effects to the changes in endogenous hormones and the effects of applied hormones, particularly gibberellins and eytokinins. The rate of expansion of the primary leaves of light-grown Phaseolus vulgaris was correlated with the gibberellin content; in etiolated leaves which did not expand, the gibberellin content was low (Wheeler, 1960). Exogenous gibberellie acid has been reported to promote expansion of leaf discs in both darkness and red light in dwarf bean (Humphries and Wheeler, 1960). Similarly,
Hormonal Control of Expansion and Greening
287
k i n e t i n will p r o m o t e the e x p a n s i o n of leaf discs of b e a n a n d there is a n i n t e r a c t i o n b e t w e e n the effects of k i n e t i n a n d red light (Powell a n d Griffiths, 1960). More recently, it has been d e m o n s t r a t e d t h a t exposure to red light leads to a r a p i d increase i n levels of endogenous gibberellins i n etiolated leaves of b a r l e y (Hordeu~n vulffare) (Reid et al., 1967). I n view of these observations on the a p p a r e n t i n v o l v e m e n t of b o t h phytochrome a n d hormones in leaf e x p a n s i o n it was decided to investigate the inter-relations of these two factors i n the expansion a n d greening of etiolated w h e a t leaves.
Materials and Methods Wheat seeds (var. "Rothwell Sprite") were grown in the dark in moist vermiculite for 7 days at 25~ C. By this time the seedlings were about 15 cm high and the first leaf, which was tightly rolled, extended 9 cm beyond the eoleoptfle. For the majority of the studies on unrolling and greening, the tip 1 cm of the leaf was excised and discarded, then the adjacent 3 cm section was excised and used. All cuttings were performed under a green safelight. For the treatments leaf segments were floated in 10 ml test solutions contained in Petri dishes. When exposure to red light was required, the dishes were placed for 5 minutes under red fluorescent tubes, screened by red "Plexiglass", giving an intensity of 4.2 • 10-scals. cm-2 min-1 (total energy). Leaf unrolling was measured by placing the leaf sections on clear plexiglas in a X4 enlarger and measuring the width of the projected image. The sections were normally measured 24 hours after exposure to red light or the application of hormones. Chlorophyll determinations were performed by extracting 10 sections with 10 ml of boiling 80% ethanol for 5 minutes and measuring the O.D. at 665 m~z. Extraction and estimation of gibberellin-likeactivity was carried out as follows. Leaf sections from 7-day old, dark-grown wheat seedlings were cut and exposed to red light for five minutes. After the required incubation period in the dark at 25~ the sections were immersed in ice cold 80% aqueous methanol and immediately subjected to a period of vacuum infiltration. The sections were then homogenised in the cold methanol. Following an extraction period of 24 hours at 1--2 ~ C, the cell debris was removed by filtration and the filtrate evaporated in vacuo at 25~ C until only the aqueous phase remained. This was partioned first at pH 7.5 and then at pH 2.5 with ethyl acetate. The acidic ethyl a~cetate fraction was reduced to dryness, redissolved in a small volume of absolute methanol and an amount equivalent to 8.0 g fresh weight was strip loaded on to Whatman No. 1 chromatography paper. The chromatogram was developed in isopropanol: ammonia:water (10:1 : 1 v/v/v) and divided into ten sections corresponding to l~f's 0.1--1.0. Eluates from the chromatogram segments were assayed using the lettuce hypocotyl test described by Frankland and Wareing (1960).
Results I n a g r e e m e n t with the report of Virgin (1962) it was f o u n d (Table 1) t h a t the unrolling of w h e a t leaves was s t i m u l a t e d b y light a n d t h a t brief exposures to red light followed b y darkness were sufficient to elicit
288
L. Beevers, B. Loveys, J. A. Pearson and P. F. Wareing:
the response. On the segments treated with A B A was effective at the response to A B A taneous applicafSon of unrolling.
other hand, it was found t h a t illuminated leaf solutions of abscisic acid (ABA) failed to unroll. concentrations as low as 0.1 ppm. Furthermore, appeared to be particularly rapid since simulA B A and red light exposure resulted in decreased
Table 1. Effect o/ abscisic acid on unrolling o/etiolated wheat leave8 Treatment
Leaf width (mm X 4)
Water (dark)
5.274- 0.18
Water (5 rain red light)
6.62 =f:0.21
0.01 ppm 0.1 ppm 1.0 ppm 10 ppm
6.20• 0.23 5.30-4- 0.17 5.45=~ 0.12 4.80 • 0.15
]
[ ABA following 5 min red light
Leaf sections were exposed to 5 min red light, then the ABA was added and the sections were allowed to remain in the dark for 24 hours before the leaf width was measured. Gibberellic acid (GAs) and kinetin both stimulated leaf segments to unroll in the dark (Table 2), although the extent of unrolling was n o t so great as in the light. Table 2. Effects o] gibberellie acid and kinetin on unrolling o] wheat teal segments in
the dark. (Lea/width in mm • 4) Hormone concentration (ppm)
GA~ Kinetin
0
2
5
10
20
6.35 5.17
7.9 5.72
8.40 6.47
7.85 6.93
-6.38
The effects of the hormones on the development of chlorophyll in etiolated leaves was studied. I t has been reported Virgin (1961) t h a t the greening process in wheat leaves exposed to continuous illumination shows an initial burst of chlorophyll production followed b y a lag phase of 3 4 hours which is, in turn, followed b y a period of continued synthesis. The lag phase can be eliminated if the leaves are given a brief illumination with red light followed b y subsequent, incubation in the dark;
Hormonal Control of Expansion and Greening
289
IL.S.D. 1%1.0 0,9 0.8 O.7 9_ E 0,6 Lt~ t.O
,.o 0.5 d 0.4 0.3 0.2 0.1 0
89
i
6
8
10
12
lh
Hours after transfer to white light
Fig. 1. Effect of gibberellic acid, khletin and abscisic acid on lag phase of chlorophyll formation in wheat leaves. 4 cm leaf sections incubated in the indieated solutions for 6 hours in the dark at 25~ C. Sections were then washed, placed in distilled water and incubated in continuous white light at 20~ C. x .x 30 rain red light given at beginning of dark incubation; 9 9 Dark control; o - - o 10ppm kinetin; 9 9 10ppmGA3;. ~ 10ppm ABA on transfer to the light synthesis and accumulation of chlorophyll begin immediately. I n attempts to replace the effect of red light in overcoming the lag phase, leaf segments were incubated in the dark in GA or kinetin for 6 hours. They were then removed from the hormones, washed, and placed in petri dishes containing water and exposed to continuous illumination. Other segments were exposed to 30 minutes of red light, without added hormones, at the beginning of the experiment, and after a further 51/2 hours in darkness they were transferred to white light. The accumulation of chlorophyll during illumination with white light was measured for all treatments (Fig. 1). The leaf segments pre-treated with red light showed the most rapid accumulation of chlorophyll. The accumulation of chlorophyll in the kinetin treated leaf segments was markedly more rapid than in the water controls, but pre-treatment with GAa had little effect on subsequent greening. Thus pre-treatment with kinetin appeared to substitute for red light in reducing the lag-phase in chlorophyll formation, but GAa appeared to be relatively ineffective. ABA leaf segments remained tightly
5o]
290
L. Beevers, B. Loveys, J. A. Pearson and P. F. Wareing:
4.0
2.0
8.ol
g701t
A
6
C
B
11o 6
R.f.
11o 6
i0
R.f.
I
t I 2.0J
r~ D R.f.
E
~O ol
R.f.
F
~:0
0 II
R.f.
11b
G 0
R.f.
s
Fig. 2A--G. Effect of red light on endogenous gibberellin levels in etiolated wheat leaves. After partition by paper chromatography the extracts were tested for gibberellin activity by the lettuce hypocotyl test. Extract from 8 g fresh weight of leaf loaded per ehromatogram. Treatments: A Dark; B--G Exposed to 5 min red light followed by 0, 5, 10, 15, 30 and 60 minutes of dark respectively
rolled and greened only slowly. This retarded chlorophyll accumulation might be due to the limited exposure of leaf surface to light in the ABA-treated segments. However, we have observed, and Mercer and Pughe (1969) have reported, a similar inhibition of chlorophyll formation by ABA in unrolled maize leaves. Thus, it appears t h a t chlorophyll formation m a y be impeded b y ABA treatment. The observations that GA 3 and kinetin could replace the effect of red light on unrolling m a y indicate t h a t the effect of red light is brought about by changes in the levels of endogenous hormones. Reid et al. (1967) reported ~hat exposure of barley leaves to red light resulted in a rapid and marked increase in gibberellin levels. I n analyses of the gibberellin content of wheat leaf segments exposed to light, it was found that there was a similar rapid increase in gibberellin levels following illumination (Fig. 2). Subsequently, the gibberellin level declined rapidly. The effect of the red light on gibberellin levels was mfllified if it was immediately followed by far-red. Since red light leads to increased gibberellin levels in the leaves, it appeared possible that the inhibition of unrolling by ABA might be mediated through its effect on endogenous gibberellin levels, and it was, indeed, found t h a t treatment with ABA inhibited the characteristic increase in gibberellins in response to red light (Fig. 3).
Hormonal Control of Expansion and Greening
291
10.0' E
9.0
E 8.0' '- 7.0 ~c~6,0` 5.0' o z~.o,
:~: 3.0' 2.0
A
c
B
R.f,
R,f.
6
Rs
i0
Fig. 3A--C. Effects of abscisic acid on the increase in gibberellins following exposure to red light. Treatments: A Dark; B 5 rain red light followed by 10 rain dark; C as for B but with 2 hours pre-treatment with ABA (10 Fg/ml). Extract from 8 g fresh weight of leaf loaded per chromatogram
7.0 84
If;
I
],sol,
'-,4" X
"41
E6.0 "ID
,~5.0 -0
{v-c-.f
-g
4..0 O1:0
2:5
5:0
10:0
Parts per million GA3 Fig. 4. Effect of gibberellie acid, in presence of various eoncentrations of
abscisic acid, on unrolling of wheat leaves. 9 0ppm ABA; ~ 1 ppm ABA; o 2.5 ppm ABA; 95.0 ppm ABA; x 10.0 ppm ABA If the only effect of A B A in preventing light-stimulated leaf unrolling was to inhibit gibberellin production, then the inhibitory effect of A B A should be overcome b y the simultaneous application of gibberellin. I t was found, however, t h a t the effects of A B A on leaf unrolling could not be overcome b y GA a (Fig. 4). Thus it appears t h a t A B A inhibits the unrolling process in some manner, other t h a n b y inhibiting the light
292
L. Beevers, B. Loveys, J. A. Pearson and P. F. Wareing:
stimulated production of GA. Further evidence in support of this latter conclusion is provided by the results of an experiment in which leaf sections were transferred to ABA solutions at various times following exposure to red light (Table 3). I t is evident that the application of ABA can be delayed for at least 150 minutes following exposure to red light and leaf unrolling is still prevented. In the intervening period between illumination and ABA application the build up of gibberellin and its disappearance would have already occurred (Fig. 1). Thus, it would appear that ABA has an inhibitory effect on some other step in the processes leading to leaf unrolling, in addition to its effect on gibberellin levels. Table 3. Inhibition o] lea] unrolling by A B A (lO ppm) /otlowing a 5 rain red light treatment (Lea/width in mm • 4, measured 2d hours after exposure)
Time of transfer to ABA after exposure to red light
Leaf width (ram • 4)
0 30 min 90 rain 150 rain 270 rain No ABA Dark control
4.05 4.33 4.06 4.90 5.60 7.00 4.10
Discussion The results of the experiments described above show that the processes leading to unrolling in etiolated wheat leaves is under phytochrome control and that both gibberellin and kinetin will stimulate unrolling in the absence of red light. The dramatic increases in the levels of acidic gibberelJins observed after only 5 minutes exposure to red light suggest that the effect of the latter on unrolling may be brought about by changes in the endogenous gibberellin levels. Some other evidence supports the view that red light effects may be mediated by increases in gibberellin levels (Brian, 1958; KShler and Lang, 1963; KShler, 1966). Conversely, the red light requirement for germination of certain seeds can be replaced by supplying exogenous gibberellin (Kahn, 1960). Reid et al. (1967) reported that the rapid rise in gibberellin levels in barley leaves following exposure to red light is inhibited by CCC. Since CCC is generally held to inhibit gibberellin biosynthesis, it would appear that red light stimulates gibberellin biosynthesis. However, the very rapid increase in gibb erellin levels following exposure to red light
ttormonal Control of Expansion and Greening
293
a n d t h e e q u a l l y r a p i d s u b s e q u e n t decline in gibberellin levels w o u l d seem to i n d i c a t e a high r a t e of gibberellin " t u r n o v e r " , for which t h e r e is little evidence f r o m o t h e r studies on gibberellin m e t a b o l i s m . A l t e r n a t i v e l y i t m a y be suggested t h a t t h e changes in gibberellin level in response to r e d light m a y i n d i c a t e t h e release of free gibberellin from a n i n a c t i v e or b o u n d f o r m a n d s u b s e q u e n t reconversion b a c k to t h e i n a c t i v e form. Thus, i t is possible t h a t t h e i n h i b i t o r y effect of A B A on t h e b u i l d u p of gibberellin levels after i r r a d i a t i o n with r e d light m a y n o t be on gibberellin biosynthesis b u t on its release from t h e i n a c t i v e form. T r e a t m e n t w i t h A B A has been f o u n d to l e a d to r e d u c e d gibberellin levels in e t i o l a t e d maize shoots (Wareing et al., 1969).
References Brian, P. W. : Role of gibberellinqike hormones in regulation of plant growth and flowering. ~ature (Lend.) 181, 1122--1123 (1958). Downs, J. : Photo-reversibility of leaf and hypocotyl elongation of dark grown red kidney bean seedlings. Plant Physiol. 30, 468-473 (1955). Frankiand, B., Wareing, P . F . : Effect of gibberellic acid on hypocotyl growth of lettuce seedlings. ~atnre (Lend.) 185, 255--256 (1960). Humphries, E. C., Wheeler, A . W . : The effects of kinetin, gibberellic acid, and light on expansion and cell division in leaf discs of dwarf bean (Phaseotus vulgaris). J. exp. Bet. 11, 81--85 (1960). Kahn, A. : Promotion of lettuce seed germination by gibberellin. Plant Physiol. 35, 333--339 (1960). Klein, W . H . , Price, L., ~itrakos, K.: Light stimulated starch degradation in plastids and leaf morphogenesis. Photochem. and Photobiol. 2, 233--240 (1963). K6hler, D.: Ver/~nderungen des GibberellingehMtes yon SMatsamen naeh Belichtung. Planta (BEE.) 70, 4 2 ~ 5 (1966). - - L a n g , A.: Evidence for substances in higher plants interfering with the response of dwarf peas to gibberellin. Plant Physiol. 38, 555--560 (1963). Liverman, J . L . , Johnson, M.P., Start, L. : Reversible photoreaction controlling expansion of etiolated bean leaf discs. Science 121, 440-441 (1955). Mercer, E. J., Pughe, J. E. : The effects of abscisic acid on the biosynthesis of isoprenoid compounds in maize. Phytochemistry 8, 115--122 (1969). Parker, M.W., ttendricks, S.B., Borthwiek, M.A., Went, F . W . : Spectral sensitivities of leaf and stem growth of etiolated pea seedlings and their similarity to action spectra for photoperiodism. Amer. J. Bet. 86, 194--204 (1949). Powell, R.D., Griffiths, M. M. : Some anatomical effects of kinetin and red light on disks of bean leaves. Plant Physiol. 85, 273--275 (1960). Reid, D.M., Clements, J.B., Carr, D . J . : Red light induction of gibberellin synthesis in leaves. Nature (Lend.) 217, 580--582 (1967). Virgin, I-I. I. : Action spectrum for the elimination of the lag-phase in chlorophyll formation in previously dark grown leaves of wheat. Physiol. Plant. (Copenh.) 14, 439--452 (1961). - - L i g h t induced unfolding of the grass leaf. Physiol. Plant. (Copenh.) 15, 380--389 (1962).
294
L. Beevers, et. al. : Hormonal Control of Expansion and Greening
Wareing, P. F., Good, J., Manuel, J. : Some possible physiological roles of abscisie acid. Proe. 6th Conference in Plant Growth Substances. Ottawa: Runge Press 1969. Wheeler, A. W. : Changes in leaf-growth substance in cotyledons and primary leaves during the growth of dwarf bean seedlings. J. exp. Bot. 11, 217---226 (1960). Leonard Beevers Department of Horticulture Univ. of Illinois Urbana, Illinois 61801 U.S.A.
Prof. P. F. Wareing Botany Department Univ. College of Wales Aberystwyth, Wales (U.K.)
Phytochrome and Hormonal Control of Expansion and Greening of Etiolated Wheat Leaves Lro~A~n B r r w ~ s , B. L o w Y s , J. A. P~,~_~so~ and P. F. WAi~I~G Botany Department, University College of Wales, Aberystwyth Received Sepember 19/November 10, 1969
Summary. UIrrolling of etiolated wheat leaf segments is stimulated by short periods of exposure to red light. Both gibberellic acid and kinetin will stimulate unrolling in the dark, whereas abscisie acid (ABA) inhibits the unrolling response to these two hormones and to red light. Exposure to 5 minutes of red light leads to a rapid increase in endogenous gibberellin levels in etiolated wheat leaves, and this increase is followed by a rapid decline. Pre-treatment with ABA inhibits the increase in gibberellin levels in response to red light, but the inhibitory effect of ABA on unrolling cannot be ascribed only to its effect on gibberellin levels. Pre-treatment with red light reduces the lag-phase in chlorophyll development when wheat leaf segments are subsequently exposed to white light; the effect of red light may be replaced by pre-treatment with kinetin, but gibberellic acid is relatively ineffective in this respect. Introduction I t has long been known t h a t leaf expansion of etiolated leaves is promoted b y light, and Parker et al. (1949) showed t h a t red light was the most effective spectral region stimulating leaf growth. I t was later demonstrated t h a t the effect of red light can be reversed by far-red light (Downs, 1955; Liverman et al., 1955), thus indicating that the phytochrome system is involved in normal leaf expansion. I n studies on wheat leaves, Virgin (1962) demonstrated that both the greening process and the leaf unrolling characteristic of graminaceous species is stimulated b y red light and reversed b y far-red light. Klein et al. (1963) have similarly demonstrated an involvement of the phytochrome system in controlling the unrolling of leaves of maize. Attempts have been made to relate these radiation effects to the changes in endogenous hormones and the effects of applied hormones, particularly gibberellins and eytokinins. The rate of expansion of the primary leaves of light-grown Phaseolus vulgaris was correlated with the gibberellin content; in etiolated leaves which did not expand, the gibberellin content was low (Wheeler, 1960). Exogenous gibberellie acid has been reported to promote expansion of leaf discs in both darkness and red light in dwarf bean (Humphries and Wheeler, 1960). Similarly,
Hormonal Control of Expansion and Greening
287
k i n e t i n will p r o m o t e the e x p a n s i o n of leaf discs of b e a n a n d there is a n i n t e r a c t i o n b e t w e e n the effects of k i n e t i n a n d red light (Powell a n d Griffiths, 1960). More recently, it has been d e m o n s t r a t e d t h a t exposure to red light leads to a r a p i d increase i n levels of endogenous gibberellins i n etiolated leaves of b a r l e y (Hordeu~n vulffare) (Reid et al., 1967). I n view of these observations on the a p p a r e n t i n v o l v e m e n t of b o t h phytochrome a n d hormones in leaf e x p a n s i o n it was decided to investigate the inter-relations of these two factors i n the expansion a n d greening of etiolated w h e a t leaves.
Materials and Methods Wheat seeds (var. "Rothwell Sprite") were grown in the dark in moist vermiculite for 7 days at 25~ C. By this time the seedlings were about 15 cm high and the first leaf, which was tightly rolled, extended 9 cm beyond the eoleoptfle. For the majority of the studies on unrolling and greening, the tip 1 cm of the leaf was excised and discarded, then the adjacent 3 cm section was excised and used. All cuttings were performed under a green safelight. For the treatments leaf segments were floated in 10 ml test solutions contained in Petri dishes. When exposure to red light was required, the dishes were placed for 5 minutes under red fluorescent tubes, screened by red "Plexiglass", giving an intensity of 4.2 • 10-scals. cm-2 min-1 (total energy). Leaf unrolling was measured by placing the leaf sections on clear plexiglas in a X4 enlarger and measuring the width of the projected image. The sections were normally measured 24 hours after exposure to red light or the application of hormones. Chlorophyll determinations were performed by extracting 10 sections with 10 ml of boiling 80% ethanol for 5 minutes and measuring the O.D. at 665 m~z. Extraction and estimation of gibberellin-likeactivity was carried out as follows. Leaf sections from 7-day old, dark-grown wheat seedlings were cut and exposed to red light for five minutes. After the required incubation period in the dark at 25~ the sections were immersed in ice cold 80% aqueous methanol and immediately subjected to a period of vacuum infiltration. The sections were then homogenised in the cold methanol. Following an extraction period of 24 hours at 1--2 ~ C, the cell debris was removed by filtration and the filtrate evaporated in vacuo at 25~ C until only the aqueous phase remained. This was partioned first at pH 7.5 and then at pH 2.5 with ethyl acetate. The acidic ethyl a~cetate fraction was reduced to dryness, redissolved in a small volume of absolute methanol and an amount equivalent to 8.0 g fresh weight was strip loaded on to Whatman No. 1 chromatography paper. The chromatogram was developed in isopropanol: ammonia:water (10:1 : 1 v/v/v) and divided into ten sections corresponding to l~f's 0.1--1.0. Eluates from the chromatogram segments were assayed using the lettuce hypocotyl test described by Frankland and Wareing (1960).
Results I n a g r e e m e n t with the report of Virgin (1962) it was f o u n d (Table 1) t h a t the unrolling of w h e a t leaves was s t i m u l a t e d b y light a n d t h a t brief exposures to red light followed b y darkness were sufficient to elicit
288
L. Beevers, B. Loveys, J. A. Pearson and P. F. Wareing:
the response. On the segments treated with A B A was effective at the response to A B A taneous applicafSon of unrolling.
other hand, it was found t h a t illuminated leaf solutions of abscisic acid (ABA) failed to unroll. concentrations as low as 0.1 ppm. Furthermore, appeared to be particularly rapid since simulA B A and red light exposure resulted in decreased
Table 1. Effect o/ abscisic acid on unrolling o/etiolated wheat leave8 Treatment
Leaf width (mm X 4)
Water (dark)
5.274- 0.18
Water (5 rain red light)
6.62 =f:0.21
0.01 ppm 0.1 ppm 1.0 ppm 10 ppm
6.20• 0.23 5.30-4- 0.17 5.45=~ 0.12 4.80 • 0.15
]
[ ABA following 5 min red light
Leaf sections were exposed to 5 min red light, then the ABA was added and the sections were allowed to remain in the dark for 24 hours before the leaf width was measured. Gibberellic acid (GAs) and kinetin both stimulated leaf segments to unroll in the dark (Table 2), although the extent of unrolling was n o t so great as in the light. Table 2. Effects o] gibberellie acid and kinetin on unrolling o] wheat teal segments in
the dark. (Lea/width in mm • 4) Hormone concentration (ppm)
GA~ Kinetin
0
2
5
10
20
6.35 5.17
7.9 5.72
8.40 6.47
7.85 6.93
-6.38
The effects of the hormones on the development of chlorophyll in etiolated leaves was studied. I t has been reported Virgin (1961) t h a t the greening process in wheat leaves exposed to continuous illumination shows an initial burst of chlorophyll production followed b y a lag phase of 3 4 hours which is, in turn, followed b y a period of continued synthesis. The lag phase can be eliminated if the leaves are given a brief illumination with red light followed b y subsequent, incubation in the dark;
Hormonal Control of Expansion and Greening
289
IL.S.D. 1%1.0 0,9 0.8 O.7 9_ E 0,6 Lt~ t.O
,.o 0.5 d 0.4 0.3 0.2 0.1 0
89
i
6
8
10
12
lh
Hours after transfer to white light
Fig. 1. Effect of gibberellic acid, khletin and abscisic acid on lag phase of chlorophyll formation in wheat leaves. 4 cm leaf sections incubated in the indieated solutions for 6 hours in the dark at 25~ C. Sections were then washed, placed in distilled water and incubated in continuous white light at 20~ C. x .x 30 rain red light given at beginning of dark incubation; 9 9 Dark control; o - - o 10ppm kinetin; 9 9 10ppmGA3;. ~ 10ppm ABA on transfer to the light synthesis and accumulation of chlorophyll begin immediately. I n attempts to replace the effect of red light in overcoming the lag phase, leaf segments were incubated in the dark in GA or kinetin for 6 hours. They were then removed from the hormones, washed, and placed in petri dishes containing water and exposed to continuous illumination. Other segments were exposed to 30 minutes of red light, without added hormones, at the beginning of the experiment, and after a further 51/2 hours in darkness they were transferred to white light. The accumulation of chlorophyll during illumination with white light was measured for all treatments (Fig. 1). The leaf segments pre-treated with red light showed the most rapid accumulation of chlorophyll. The accumulation of chlorophyll in the kinetin treated leaf segments was markedly more rapid than in the water controls, but pre-treatment with GAa had little effect on subsequent greening. Thus pre-treatment with kinetin appeared to substitute for red light in reducing the lag-phase in chlorophyll formation, but GAa appeared to be relatively ineffective. ABA leaf segments remained tightly
5o]
290
L. Beevers, B. Loveys, J. A. Pearson and P. F. Wareing:
4.0
2.0
8.ol
g701t
A
6
C
B
11o 6
R.f.
11o 6
i0
R.f.
I
t I 2.0J
r~ D R.f.
E
~O ol
R.f.
F
~:0
0 II
R.f.
11b
G 0
R.f.
s
Fig. 2A--G. Effect of red light on endogenous gibberellin levels in etiolated wheat leaves. After partition by paper chromatography the extracts were tested for gibberellin activity by the lettuce hypocotyl test. Extract from 8 g fresh weight of leaf loaded per ehromatogram. Treatments: A Dark; B--G Exposed to 5 min red light followed by 0, 5, 10, 15, 30 and 60 minutes of dark respectively
rolled and greened only slowly. This retarded chlorophyll accumulation might be due to the limited exposure of leaf surface to light in the ABA-treated segments. However, we have observed, and Mercer and Pughe (1969) have reported, a similar inhibition of chlorophyll formation by ABA in unrolled maize leaves. Thus, it appears t h a t chlorophyll formation m a y be impeded b y ABA treatment. The observations that GA 3 and kinetin could replace the effect of red light on unrolling m a y indicate t h a t the effect of red light is brought about by changes in the levels of endogenous hormones. Reid et al. (1967) reported ~hat exposure of barley leaves to red light resulted in a rapid and marked increase in gibberellin levels. I n analyses of the gibberellin content of wheat leaf segments exposed to light, it was found that there was a similar rapid increase in gibberellin levels following illumination (Fig. 2). Subsequently, the gibberellin level declined rapidly. The effect of the red light on gibberellin levels was mfllified if it was immediately followed by far-red. Since red light leads to increased gibberellin levels in the leaves, it appeared possible that the inhibition of unrolling by ABA might be mediated through its effect on endogenous gibberellin levels, and it was, indeed, found t h a t treatment with ABA inhibited the characteristic increase in gibberellins in response to red light (Fig. 3).
Hormonal Control of Expansion and Greening
291
10.0' E
9.0
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:~: 3.0' 2.0
A
c
B
R.f,
R,f.
6
Rs
i0
Fig. 3A--C. Effects of abscisic acid on the increase in gibberellins following exposure to red light. Treatments: A Dark; B 5 rain red light followed by 10 rain dark; C as for B but with 2 hours pre-treatment with ABA (10 Fg/ml). Extract from 8 g fresh weight of leaf loaded per chromatogram
7.0 84
If;
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],sol,
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Parts per million GA3 Fig. 4. Effect of gibberellie acid, in presence of various eoncentrations of
abscisic acid, on unrolling of wheat leaves. 9 0ppm ABA; ~ 1 ppm ABA; o 2.5 ppm ABA; 95.0 ppm ABA; x 10.0 ppm ABA If the only effect of A B A in preventing light-stimulated leaf unrolling was to inhibit gibberellin production, then the inhibitory effect of A B A should be overcome b y the simultaneous application of gibberellin. I t was found, however, t h a t the effects of A B A on leaf unrolling could not be overcome b y GA a (Fig. 4). Thus it appears t h a t A B A inhibits the unrolling process in some manner, other t h a n b y inhibiting the light
292
L. Beevers, B. Loveys, J. A. Pearson and P. F. Wareing:
stimulated production of GA. Further evidence in support of this latter conclusion is provided by the results of an experiment in which leaf sections were transferred to ABA solutions at various times following exposure to red light (Table 3). I t is evident that the application of ABA can be delayed for at least 150 minutes following exposure to red light and leaf unrolling is still prevented. In the intervening period between illumination and ABA application the build up of gibberellin and its disappearance would have already occurred (Fig. 1). Thus, it would appear that ABA has an inhibitory effect on some other step in the processes leading to leaf unrolling, in addition to its effect on gibberellin levels. Table 3. Inhibition o] lea] unrolling by A B A (lO ppm) /otlowing a 5 rain red light treatment (Lea/width in mm • 4, measured 2d hours after exposure)
Time of transfer to ABA after exposure to red light
Leaf width (ram • 4)
0 30 min 90 rain 150 rain 270 rain No ABA Dark control
4.05 4.33 4.06 4.90 5.60 7.00 4.10
Discussion The results of the experiments described above show that the processes leading to unrolling in etiolated wheat leaves is under phytochrome control and that both gibberellin and kinetin will stimulate unrolling in the absence of red light. The dramatic increases in the levels of acidic gibberelJins observed after only 5 minutes exposure to red light suggest that the effect of the latter on unrolling may be brought about by changes in the endogenous gibberellin levels. Some other evidence supports the view that red light effects may be mediated by increases in gibberellin levels (Brian, 1958; KShler and Lang, 1963; KShler, 1966). Conversely, the red light requirement for germination of certain seeds can be replaced by supplying exogenous gibberellin (Kahn, 1960). Reid et al. (1967) reported that the rapid rise in gibberellin levels in barley leaves following exposure to red light is inhibited by CCC. Since CCC is generally held to inhibit gibberellin biosynthesis, it would appear that red light stimulates gibberellin biosynthesis. However, the very rapid increase in gibb erellin levels following exposure to red light
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293
a n d t h e e q u a l l y r a p i d s u b s e q u e n t decline in gibberellin levels w o u l d seem to i n d i c a t e a high r a t e of gibberellin " t u r n o v e r " , for which t h e r e is little evidence f r o m o t h e r studies on gibberellin m e t a b o l i s m . A l t e r n a t i v e l y i t m a y be suggested t h a t t h e changes in gibberellin level in response to r e d light m a y i n d i c a t e t h e release of free gibberellin from a n i n a c t i v e or b o u n d f o r m a n d s u b s e q u e n t reconversion b a c k to t h e i n a c t i v e form. Thus, i t is possible t h a t t h e i n h i b i t o r y effect of A B A on t h e b u i l d u p of gibberellin levels after i r r a d i a t i o n with r e d light m a y n o t be on gibberellin biosynthesis b u t on its release from t h e i n a c t i v e form. T r e a t m e n t w i t h A B A has been f o u n d to l e a d to r e d u c e d gibberellin levels in e t i o l a t e d maize shoots (Wareing et al., 1969).
References Brian, P. W. : Role of gibberellinqike hormones in regulation of plant growth and flowering. ~ature (Lend.) 181, 1122--1123 (1958). Downs, J. : Photo-reversibility of leaf and hypocotyl elongation of dark grown red kidney bean seedlings. Plant Physiol. 30, 468-473 (1955). Frankiand, B., Wareing, P . F . : Effect of gibberellic acid on hypocotyl growth of lettuce seedlings. ~atnre (Lend.) 185, 255--256 (1960). Humphries, E. C., Wheeler, A . W . : The effects of kinetin, gibberellic acid, and light on expansion and cell division in leaf discs of dwarf bean (Phaseotus vulgaris). J. exp. Bet. 11, 81--85 (1960). Kahn, A. : Promotion of lettuce seed germination by gibberellin. Plant Physiol. 35, 333--339 (1960). Klein, W . H . , Price, L., ~itrakos, K.: Light stimulated starch degradation in plastids and leaf morphogenesis. Photochem. and Photobiol. 2, 233--240 (1963). K6hler, D.: Ver/~nderungen des GibberellingehMtes yon SMatsamen naeh Belichtung. Planta (BEE.) 70, 4 2 ~ 5 (1966). - - L a n g , A.: Evidence for substances in higher plants interfering with the response of dwarf peas to gibberellin. Plant Physiol. 38, 555--560 (1963). Liverman, J . L . , Johnson, M.P., Start, L. : Reversible photoreaction controlling expansion of etiolated bean leaf discs. Science 121, 440-441 (1955). Mercer, E. J., Pughe, J. E. : The effects of abscisic acid on the biosynthesis of isoprenoid compounds in maize. Phytochemistry 8, 115--122 (1969). Parker, M.W., ttendricks, S.B., Borthwiek, M.A., Went, F . W . : Spectral sensitivities of leaf and stem growth of etiolated pea seedlings and their similarity to action spectra for photoperiodism. Amer. J. Bet. 86, 194--204 (1949). Powell, R.D., Griffiths, M. M. : Some anatomical effects of kinetin and red light on disks of bean leaves. Plant Physiol. 85, 273--275 (1960). Reid, D.M., Clements, J.B., Carr, D . J . : Red light induction of gibberellin synthesis in leaves. Nature (Lend.) 217, 580--582 (1967). Virgin, I-I. I. : Action spectrum for the elimination of the lag-phase in chlorophyll formation in previously dark grown leaves of wheat. Physiol. Plant. (Copenh.) 14, 439--452 (1961). - - L i g h t induced unfolding of the grass leaf. Physiol. Plant. (Copenh.) 15, 380--389 (1962).
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Wareing, P. F., Good, J., Manuel, J. : Some possible physiological roles of abscisie acid. Proe. 6th Conference in Plant Growth Substances. Ottawa: Runge Press 1969. Wheeler, A. W. : Changes in leaf-growth substance in cotyledons and primary leaves during the growth of dwarf bean seedlings. J. exp. Bot. 11, 217---226 (1960). Leonard Beevers Department of Horticulture Univ. of Illinois Urbana, Illinois 61801 U.S.A.
Prof. P. F. Wareing Botany Department Univ. College of Wales Aberystwyth, Wales (U.K.)
