Planta (Berl.) 75, 291--308 (1967)

Gibberellic Acid and the Light Inhibition of Stem Elongation DA]?III~IE VINCE University of Reading Horticultural Research Laboratories, Shinfield Grange, Shinfield, Berkshire Received March 28, 1967

Summary. The ability of gibberellie acid (GAs) to prevent the light inhibition of stem elongation in peas was examined for several varieties under a wide range of irradiation conditions. A saturating dose of GA S largely prevented the inhibitory effect of red light on total stem height in " D u k e of Albany" (tall), " A l a s k a " (medium) and " M e t e o r " (dwarf) although a small, but statistically significant, effect persisted in all varieties after 3 days of light. The growth of the second internode was, however, markedly inhibited by red light even with a saturating dose of GA S. With gibberellin there was no difference between the effects of continuous red light and 15 minutes per day on height but the second internode was much shorter in the former treatment. The number of internodes present was the same in both cases and, therefore, the upper internodes in continuous light were as long or longer than in the 15-minute treatment. The number of internodes was only slightly fewer in darkness than in light so that, with GA S, the effect of red light was transient and only the growth of the lower internodes was inhibited. Without GA S overall height was less in both red light treatments than in darkness for all three varieties. In blue light, on the other hand, there was no difference depending on whether height or internode length is considered, and even with a saturating dose of GA S the growth rate remained depressed in continuous blue light. There was, however, some interaction between blue light and GAS. Red/far-red reversal experiments showed that in the varieties "Alaska" and " D u k e of Albany" the far-red stimulation of elongation persisted in the presence of a saturating dose of GA 3 while for the dwarf variety " M e t e o r " there was a significant interaction between far-red and GA S. At least a quantitative difference was found between tall and dwarf peas in their response to light. Tall varieties showed a much greater effect of a prolonged exposure to blue and ~ smaller effect of a short exposure to red than dwarf varieties. Increasing the duration of exposure to red increasingly inhibited the growth of tall varieties. The medium variety "Alaska" grew to approximately the same height in continuous red and blue light. Introduction O n e of t h e c o n t r o v e r s i a l q u e s t i o n s a b o u t l i g h t c o n c e r n s t h e possible r e l a t i o n s h i p of g i b b e r e l l i n s w i t h l i g h t effects o n s t e m g r o w t h . I n s o m e species (e.g. Sinapis alba, MOH~ a n d AI'PUHN, 1962; NWAOHU~:U a n d LOCl~_rrAI~T, 1964) t h e r e is no i n t e r a c t i o n b e t w e e n t h e effects of a p p l i e d g i b b e r e l l i c a c i d (GAs) a n d l i g h t : t h i s f i n d i n g h a s b e e n i n t e r p r e t e d t o 21 Planta (Berl.), Bd. 75

292

D. VI~cm

mean that no close relationship exists between light and gibberellins; however, as LOC~:H~T and DEAL (1960) have shown for Cucurbita pepo and Cucumis sativa with GA4, an interaction between light and another gibberellin may occur when there is none with GA 8. Examples of statistically valid interactions between light and applied GA3 are numerous. In some cases the interaction is complete, plants with saturating doses of GA8 showing no significant inhibition of stem elongation by light; this is so in Perilla ocymoides (LoNA and BoccHI, 1956) and for total height in Pisum sativum (LocKI~)mT, 1956) and Phaseolus vulgaris (LocKHART, 1958, 1961). In others GA 8 does not completely prevent the light inhibition of elongation, as in internode growth in Pisum sativum (LocKHART, 1956; SAL~ and VINci, 1960; SIMPSON and W ~ , 1961) and hypoeotyl elongation in Lactuca sativa "Grand Rapids" (FRA~I~A~D and WARE~G, 1960), Lycopersicon esculentum " P o t e n t a t e " (PEGG, 1962) and Phaseolus aureus (~qWACHUKUand LOC~gART, ]964). These observations led LOCKHART(1961) to suggest that light affects the rate of stem elongation by an effect on endogenous gibberellins : he further suggested t h a t the apparently incomplete reversal by GA a reported by SALw and V ~ c ~ , and others, was due to the fact that these authors measured final lengths of internodes rather than total height. LOCKHART argued that, if the rate of node formation is affected b y light, then only measurements of total stem height during the linear phase of growth will give a true picture of the effect of GAa on elongation rate. The problem is whether any of the postulated photomorphogenetic systems can be specifically related to an effect on endogenous gibberellins. I t is generally accepted that at least two light-sensitive systems can be detected in the control of stem elongation; one of these is the low energy red/far-red reversible reaction of phytochrome, the other becomes important under conditions of prolonged irradiation (MooR, 1957; S~mE and V ~ c ~ , 1959; M~IJ~R, 1959; Vrz~cw, 1964) and has been called the "high-energy reaction". The latter has peaks of action in blue and frequently also in far-red; the far-red peak may be a variant of phytochrome action (GRILL and V ~ c ~ , 1965; H~_RT~A~, 1966) but the blue effect appears to depend on another pigment system which has not yet been identified (V~cE and G ~ , 1966). So far gibberellins have not been shown to be directly implicated in any one of these systems (for a further discussion see V ~ c E , 1964). In Pisum sativum an interaction between GAa and light has been clearly demonstrated. There is also a difference between the responses of tall and dwarf peas both to light and GA3 : dwarf varieties are particularly sensitive to the application of GAa, and the response is much greater in the light than in darkness. The difference in height between tall and dwarf varieties is smaller in the dark, the growth of dwarfs being in-

Gibberellic Acid and the Light Inhibition of Stem Elongation

293

h i b i t e d more b y light. The two types also respond differently to wavelength. I n dwarfs there is a large i n h i b i t i o n of growth with a short exposure to red light and, even with long light periods, p l a n t s are shorter i n red t h a n i n b l u e : i n tall peas growth is i n h i b i t e d more i n blue t h a n i n red when i r r a d i a t i o n periods of several hours d u r a t i o n are given (S~]~ a n d VI~c]~, 1960). Recently, however, GORT~R (1964) was u n a b l e to demonstrate a n y difference i n spectral sensitivity b e t w e e n the tall v a r i e t y " A l a s k a " a n d two dwarf varieties w h e n t h e y were grown i n c o n t i n u o u s light of high i n t e n s i t y ; u n f o r t u n a t e l y in these experiments the light sources were e q u a t e d on the basis of energy r a t h e r t h a n q u a n t u m intensity. I n the experiments presented here a detailed s t u d y of stem elongation has been m a d e on several varieties of pea u n d e r various conditions of i r r a d i a t i o n i n a n a t t e m p t to determine whether a n association of gibberellin with a n y specific light effect on elongation could be established.

Materials and Methods The light sources were lamps with filters giving blue (400--520 nm), red (600--700 nm) or far-red ( ~ 700 nm) light (GRILL, 1965). The red and blue sources were adjusted to give equal incident quanta at bench height; the measured intensities were 9.0 Kergs cm-2 sec-1 in blue and 6.6 in red. Temperatures were maintained at 25--260 C. Seeds were sown in sand and germinated in the dark for four days before treatment. Usually a single application of gibberellic acid in 80 % ethanol was made as a 0.002 ~l drop to the apical bud of each plant, using dim green light from an 8-watt fluorescent tube wrapped in Strand no. 39 primary green "Cinemoid" filter: control plants received ethanol. The same dose given daffy in three applications gave the same response as a single dose. (In one experiment [Fig. 3] a spray containing 80 ppm GAa in 20% ethanol was given daily for 3 days, just before the beginning of each irradiation treatment). ])ark grown plants were treated with GA3 at the same time as those in the light. The irradiation treatments began within one hour after the application of GAs and were then repeated daily for 3 days; in the 24-hour treatments plants remained in continuous light for 72 hours, otherwise plants were returned to darkness for the remainder of each day. The standard irradiation treatments were 15 minutes and 24 hours of red or blue light: with the former only the red/far-red reversible reaction of phytochrome would be expected to affect elongation but, with the long period of light, both the phytochrome system and the "high-energy reaction" would operate. Prolonged exposures to far-red were not included because of the doubt as to how the effects of these are related to phytochrome action. Measurements of growth rates in the dark for plants grown with and without GAs showed that elongation in terms of total stem height was linear with time from the fifth to at least the tenth day from sowing. Plants were, therefore, usually measured on the seventh day. Unless otherwise stated five plants were measured in each treatment and the means given are from at least two separate experiments; each experiment was considered as a block in the statistical analysis. For measurements of epidermal cell lengths, pieces of epidermis were stripped from the central portion of the fully expanded second internode and mounted in an aqueous solution of brilliant cresyl blue; 20 cells were measured in each internode, a total of 100 in each treatment; the values given are the means from two 21"

294

D. VI~cE:

experiments, i.e. for 200 cells. Epidermal cell numbers were obtained by dividing internode length by the mean cell length. The varieties used in the main experiments were "Meteor", "Alaska" and "Duke of Albany"; according to SNEDDO• and SQUIBBS'(1958) height classification these are respectively dwarf (under 45 cm), medium (75--111 era) and tall (above 111 era). Results

E//ect o/Light on Height, Internode Length, and True and Apparent Plastochron I n view of the apparent discrepancy in results when total stem height and final internode lengths are taken as measurements of light effects it was necessary first to establish the effects of the standard light treatments on these growth parameters and on the plastochron interval. Fig. 1 shows the results from a single experiment with "Alaska". The major p a r t of the growth of the second internode occurred between the fifth and seventh d a y from sowing. The effect of the 15-minute light treatments on total stem height was transient; growth was inhibited immediately after the first irradiation but the growth rate again approximately equalled t h a t in darkness after one day, even though the plants continued to be irradiated daily. I n continuous light also the growth rate increased after an initial depression but not to equal the dark growth rate. I n continuous light the heights in red and blue were approximately the same but, with the 15-minute treatments, red was slightly more inhibitory than blue. The re-establishment of a growth rate equal to t h a t in the dark when short daily irradiations were used is similar to the pattern found b y LOCK~A~T and GOTTSC~AnL (1959) in continuous low intensity red; with continuous high intensity red light they also found a permanent depression of the growth rate in "Alaska". They could not, however, find any effect of a 10-minute daffy irradiation on stem height in "Alaska". As the effect of light on the number of expanded leaves and on the total leaf number is similar, light appears to affect both the rate of leaf inception at the apex and the rate of unfolding from the apical bud; the latter may, however, be only a secondary effect. Up to the seventh or eighth day all light treatments were similar, the plastoehron interval between the ninth and tenth leaf being approximately one day in the fight and 1.5 days in darkness; on the seventh d a y there was 0.7 of a leaf more present in light t h a n in the dark. Only on and after the ninth d a y was there any difference between light treatments, when continuous red light accelerated leaf inception and unfolding more t h a n the other treatments; this effect could be a photosynthetic one. Subsequently the seventh day was chosen as the date for measuring as b y then the second internode was fully expanded, the growth rate in terms of height was linear with time and characteristic for the light

Gibberellic Acid and the Ligh~ Inhibition of Stem Elongation

295

treatment, and the effect of light on leaf n u m b e r was quite small, there being no difference between the standard light treatments a n d only 0.7 leaves fewer in darkness.

,,::~

~zo

//;'

9

9

4

5

o

",, :,

-o

Re" r o ---o

.fD --- [] 75rnlnuf~ Blue L"-, continuous

_=,

9

75mlnules

o ~. _ _ . continuous

|

[ zSminutes red # blue " [ oontinuous blue

-~ ~JoF 150

5or

I

I

r

)

I

I

Fig. I. Effect of the standard light treatments on height, growth of the second internode and leaf number in "Alaska" pea. Irradiation treatments began on the fourth day after sowing and were subsequently given daily EMects of GA a on the Inhibition o[ Growth by Light These experiments were carried out in two parts. I n the first, GA a doses ranging from 0 - - 3 ~g per plant were used and in the second, doses from 3 - - 3 0 0 [zg per plant. I n both cases the varieties "Alaska", "Meteor" and " D u k e of A l b a n y " were grown and the s t a n d a r d light t r e a t m e n t s

296

D. VI~eE:

given. The results for 3--300 ~tg GA s are given in Table 1 and for 0 - - 3 ~tg GA 3 in Fig. 2. Analysis of the results from Table 1 showed no significant effect of GA 3 dose and no significant interaction between GA s and light; thus GA s was present at saturating doses for all light treatments and varieties, both for total height and internode length. The light means can, therefore, be taken as the response to fight in the presence of a saturating dose of gibberellin. Two main conclusions can be drawn from these results. Firstly, whether total height or internode length is taken, a statistically significant photoinhibition of stem elongation occurred in all varieties in the presence of saturating doses of GA a. Secondly, there is a difference between the results depending on whether internode length or total height is considered. For total height all plants were significantly shorter in fight t h a n in the dark (except 15 minutes blue and continuous red in " D u k e " ) but the only difference between fight treatments is the large depression of growth in continuous blue; this occurred in all varieties but was particularly marked in " D u k e of A l b a n y " : in no case was there a significant difference between continuous red and 15 minutes red. For internode length, however, continuous red was significantly (P = 0.05) more inhibitory than 15 minutes red for the taller varieties and almost so for " M e t e o r " (internode 2 was shorter with 24 hours than with 15 minutes red for four out of five doses of GAs). As at this stage there is no difference between light treatments in the number of leaves expanded the growth rate must have been fully re-established in red light after the expansion of the second internode; the results thus suggest t h a t with saturating doses af GA s only the growth of the second internode is inhibited more b y continuous t h a n b y 15 minutes red; this m a y be due only to a delay in transloeation of GA s as the irradiation treatments began almost immediately after GA a was applied. I t appears t h a t GA s specifically prevents an effect of red light on elongation and has less effect on a timedependent blue fight response. The results for doses of 0 - - 3 ~g GA a are given in Fig. 2. All varieties show GA a - - fight interaction curves at both wavelengths, the light inhibition being less with a saturating dose of GA a (3 ~g) than without GA s and the values in the fight approaching those in the dark as the dose is increased. However, some photoinhibition still occurred in the presence of a saturating dose of GA a, especially in continuous blue light. For both blue light treatments the relative inhibition of growth was the same for internode length and height. For red fight this is also true of the treatments without GA a but, with saturating GA s, red was more inhibitory to growth in length of the second internode t h a n to total height; growth of later internodes must, therefore, have been fully restored when GA s was present.

Gibberellic Acid and the Light Inhibition of Stem Elongation

297

Table. Effect of wave-length and duration o) light at saturating doses o/ gibbereUin Variety

Light treatment (4--6th day from sowing)

GA dose (~g per plant) 3

10

30

100

300

Mean

A. Total height (mm) Alaska

Duke of Albany

Meteor

Dark

221.6 226.6 226.3 216.9 202.7

218.84442.86"

Red, 15min. daily Red, continuous

215.1 201.6 198.0 201.3 197.6 217.9 208.0 204.5 180.9 194.7

Blue, 15min. daily Blue, continuous

201.9 197.6 211.3 205.9 187.8 183.2 195.8 196.5 177.0 175.2

202.7] 201"2~4-4 04* 200.9[ "

Dark

190.2 208.5 207.1 195.6 185.9

197.54443.14"

Red, 15min. daily Red, continuous Blue, 15min. daily Blue, continuous

178.8 177.0 184.7 186.6 182.7 181.5 192.0 185.0 183.2 191.9 190.3 193.2 197.0 190.4 189.0 140.9 140.2 151.4 140.4 120.0

182.0] 186.77_4_4.44* 192.0[ 138.6/

Dark

187.2 188.4 191.1 187.8 178.3

186.5 4442.28 *

Red, 15min. daily Red, continuous

167.5 166.9 170.1 178.7 174.9 172.9 175.5 178.6 173.3 174.6

171.6] 175"0~:~3 23*

Blue, 15min. daffy Blue, continuous

177.5 179.7 179.2 167.7 168.3 165.8 163.3 159.5 150.0 142.9

i74.5|

185.5)

"

156.3J

B. Internode 2, ]inal length (ram) Alaska

Duke of Albany

Me,or

Dark

89.6

89.9

86.4

87.8

81.9

87.14441.16"

Red, 15min. daily Red, continuous

70.2 63.2

74.5 63.2

74.6 75.5 6 6 . 1 58.4

71.7 63.7

Blue, 15min. daily Blue, continuous

79.3 63.5

7 9 . 1 7 8 . 1 74.9 61.7 6 4 . 7 59.4

74.0 62.5

73.3I 62"914-1.63 * 77.1[ 62.4)

Dark

87.0

89.5

88.3

87.4

86.6

87.84441.36*

Red, 15min. daffy Red, continuous

73.5 65.6

76.6 68.4

81.0 78.4 6 3 . 1 61.2

79.6 62.7

Blue, 15min. daily Blue, continuous

86.0 47.8

84.8 49.6

81.8 52.3

83.6 41.5

77.8] 64.2[• 1.92 83.6| 48.4]

81.6 50.4

Dark

77.8

77.0

80.4

77.2

76.4

77.7• 1.35 *

Red, 15rain. daily Red, continuous

69.7 59.2

65.3 64.9 6 2 . 6 68.8

72.4 63.0

65.0 58.7

Blue, 15min. daily Blue, continuous

71.6 58.9

8 1 . 3 72.0 5 9 . 3 58.3

72.8 56.8

77.9 51.7

67.5 I 62"5~-t-1.91 * 75.1| 57.0)

* S.E. mean.

298

D. V i n c i :

The results s u p p o r t t h e conclusion r e a c h e d f r o m t h e p r e v i o u s experim e n t t h a t G A s p r e v e n t s a n effect of r e d light on g r o w t h a n d has less effect in blue l i g h t ; nevertheless t h e G A a dose curves in blue l i g h t do show a n i n t e r a c t i o n w i t h those in darkness, g r o w t h being p r o m o t e d /./ei,.qht Red mm

Blue

Duke of a/banU

2/O 170 /3O gO 5O

Alaska

23O /8O /5O

I"

/10 7O /3O

f/e/eop

150 lIO 7O a

3O

o

o.oo3 0.03 o.3

3

o

o.oos o.os o.3

3

~.GA3 Fig. 2a and b. Effect of gibberellic acid on height and length of the second internode under the standard irradiation conditions. Plants were given 15 minutes (O ..... C)) or 24 hours ( . . ) light each day or remained in darkness (11 II). Irradiation treatments began on the 4th day and plants were measured on the 7th day from sowing. * Significant difference at P ~ 0.05 m o r e in blue light t h a n in t h e d a r k . A n earlier e x p e r i m e n t (Fig. 3) w i t h t h e v a r i e t y " D u k e of A l b a n y " grown w i t h a n d w i t h o u t G A 3 shows t h e response to v a r i o u s d u r a t i o n s of blue light given each d a y . The figures are l e n g t h s of t h e fully e x p a n d e d second a n d t h i r d i n t e r n o d e s ; measurem e n t s of h e i g h t were n o t m a d e b u t in blue light these should reflect t h e

Gibberellic Acid and the Light Inhibition of Stem Elongation

299

final internode lengths. The effect of GA3 was mainly on a relatively short duration effect of blue light; with 24 hours of blue light the effect of GAa was not much greater than in the dark but, with exposure times of 4--12 hours, there was a considerably larger effect.

Len#lh of second /nlefno#e Red

rrlli'l

Blue

Oukeofalban#

100

80 gO z/O

100

Alaska

80 8O ~0 20 8O

Me/cop

80 qO 2O

o. os o)s o.'3 3

o.o'os o..'os o'3 3 ~u.g.aA3

Fig. 2 b (for legends see 13. 298)

E//ects o/GA 3 on Cell Length and Number Measurements of epidermal cell lengths were made on plants from the 0 and 3 ~g treatments; these, together with the calculated values for the number of cells along the length of the internode, are given in Fig. 4. I t is clear that the main effect of light is to decrease cell length; the decrease in cell number was much smaller and, in " D u k e of Albany", there was no significant reduction by any light treatment. The application of GAa caused a small increase in cell number, which was more or less the

300

D. Vn~OE:

same in all light treatments, but the major effect on intcrnode length resulted from the large increase in cell elongation. I n all three varieties the effect of GA3 on cell length was greater in the light than in darkness. I n " A l a s k a " and "Meteor" only in continuous blue light were cells with GA3 significantly shorter than in the dark but, i n " Duke of Albany", they were shorter in all light treatments except 15 minutes blue. I t is clear that, although light decreases and GA3 increases the number of epidermal cells in the length of the internode, the major effect of both

.0r\

170t \~...... 100t" "--.~.

"----.

5O

D

h

hrs blueI/~h/

Fig. 3. Effect of gibberellic acid with various durations of blue light each day; variety "Duke of Albany". Irradiation treatments began on the 4th day and plants were measured on the 7th day from sowing. Plants were sprayed daily with 80 ppm GAa from 4th day to 6th day. On the ordinate, mean length of internodes 2 and 3 treatments is on cell elongation; any interaction between effects of GAs and light primarily concerns effects on cell growth rather than cell division.

The Red/Far-Red Reversible Reaction One of the critical questions concerning the interaction between gibberellin and light is whether plants given a saturating dose of GAa still show the red/~ar-red reversible reaction of phytochrome, i.e. whether there is an interaction between GA3 and the phytochrome system. LOCK~A~T (1961) showed that, in the dwarf pea "Morse's Progress no. 9", there was no red/far-red effect on stem height with saturating doses of gibberellin. For final internode lengths in "Meteor" and "Alask a " , however, far-red and GA3 effects were virtually additive (SALE and V ~ c ~ , 1960) and a similar result was obtained with excised internode sections from " A l a s k a " by HTLLMAN (1959). An experiment was, therefore, carried out with the three varieties of pea to test the response to red and far-red radiation with and without saturating doses of GAz

Gibberellic Acid and the Light Inhibition of Stem Elongation

301

(Fig. 5). As before t h e p l a n t s were t r e a t e d w i t h G A a (10 ~g p e r p l a n t ) j u s t before t h e first i r r a d i a t i o n a n d t h e i r r a d i a t i o n t r e a t m e n t s were given d a i l y for t h r e e d a y s . Two d u r a t i o n s of light were given t o see w h e t h e r f a r - r e d r e v e r s i b i l i t y persisted after several hours of i r r a d i a t i o n . Blue light was also used a n d t h e results were i n c l u d e d in t h e s t a t i s t i c a l

Cell lengh~

Cellnumber

Duke of I*

a/banff -I-

ego 4100[

r,~_]

100

']

200[ OL

o lE2~

o 152~

red

blue

01S241 red

01s blue

0

Alaska i~

h'-','--I ;-'-)-i 0L

015241 red

0752g blue

',

,

t r_j._. II

I

200[

0L

0 1524l fed

0 152g

r-,

~-,

0

blue

]+

Meteor 000

700

i

1120

s .-,

,, ,,,' ,

!

0 O 752g 0 N 2 g 0 fS2~ red blue red blue Fig. 4. Effect of gibberellic acid on epidermal cell length and number in the second internode. 0 ~ darkness, 15 = 15 minutes light, 24 = 24 hours light each day. Dotted bars - - with GAa. Experimental conditions as in Fig. 2. * Significant difference at P = 0.05

0 752q

a n a l y s i s : t h e y were n o t different from those with r e d a n d therefore are n o t given in t h e figure. A n a l y s i s of t h e results showed no significant difference in t h e response to far-red b e t w e e n t o t a l h e i g h t a n d i n t e r n o d e length, e x c e p t in " A l a s k a " where t h e far-red p r o m o t i o n of g r o w t h was significant o n l y for i n t e r n o d e l e n g t h a n d n o t for t o t a l height. T h e r e was a difference in t h e response of " M e t e o r " a n d t h e two t a l l e r varieties.

]02

D. VINCE:

I n " A l a s k a " ( i n t e r n o d e l e n g t h ) a n d " D u k e of A l b a n y " (height a n d i n t e r n o d e l e n g t h ) p l a n t s r e c e i v i n g f a r - r e d were s i g n i f i c a n t l y ( P = 0.05) l o n g e r t h a n t h o s e w i t h o u t , a n d t h e r e was n o i n t e r a c t i o n b e t w e e n f a r - r e d

tiei'gkt

mm rl-, Boo. "' sso

,#0

r,l#~ ,--,-, "'

~]

fl ,!1

I I ]

0

200

Lenjlk o# second /zfernode

fl

fl/ m~ D.k~o: ~,ib

Gibberellic acid and the light inhibition of stem elongation.

The ability of gibberellic acid (GA3) to prevent the light inhibition of stem elongation in peas was examined for several varieties under a wide range...
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