Plant Physiol.

(1971) 47, 705-708

Effect of the Hypocotyl Hook on Chlorophyll Accumulation in

Excised Cotyledons of Cucumis sativus L.
Received for publication October 22, 1970

SAMUEL I. HARDY,' PAUL A. CASTELFRANCO, AND CONSTANTIN A. REBEIZ

Department of Bontany, University of California, Davis, California 95616
ABSTRACr

Hypocotyl hooks have been shown to influence greening in

excised cucumber (Cucumis sativus) cotyledons. The properties
of the lag phase are greatly affected by the presence or absence
of the hook tissue. A 45-second light pretreatment followed by
4 hours of darkness is sufficient to remove the lag phase from
cotyledons with hooks, while hookless cotyledons require 2
hours of continuous illulmination followed by 1 hour of dark
incubation to break the lag phase. The effect of hooks on cotyledon greening is enhanced if the hooks are shielded from
light. Cutting off the hooks after lag phase removal caused a
marked decrease in chlorophyll accumulation in the cotyledons.
These observations may indicate that the hypocotyl hooks produce a substance or substances needed in the greening process,
which are translocated to the cotyledons. Indoleacetic acid,
abscisic acid, gibberellin A,, 6-benzylamino purine and 8-aminolevulinic acid do not show any activity; on the other hand,
ethylene appears to replace partialy the hypocotyl hooks.

In a previous communication (2) we reported that etiolated

cucumber cotyledons excised with full hypocotyl hook green as
rapidly as the cotyledons attached to whole etiolated seedlings,
while cotyledons excised without hook exhibit an abnormally
long lag phase with respect to chlorophyll accumulation. It was
concluded that a contribution of the hypocotyl hook is essential for the normal greening response of excised cucumber
cotyledons (2). In this article we report a series of experiments
which further describe the greening process of etiolated cucumber cotyledons excised with and without hooks.

MATERAILS AND METHODS

Indoleacetic acid and 8-aminolevulinic acid-HCl were obtained from Eastman. Abscisic acid was a gift of Dr. F. T.
Addicott. Gibberellin A. was a Sigma product; 6-benzylamino
purine was obtained from Mann Research Laboratories.
Cucumber seed (Cucumis sativus L. var Alpha green, a gift
of the Niagara Chemical Division, FMC Corporation, Modesto,
Calif.) were germinated for 4 days in the dark at 24 C and
harvested as reported previously (2). The etiolated cotyledons
were excised as described earlier (2). The plant material was
incubated with mild agitation at 28 C in 50-ml beakers containing 2.0 ml of H110. Light incubations were carried out under
240 ft-c of white fluorescent light from a G.E. Cool White
lamp. Short term light treatments were performed in the
darkroom with the same light source attached to an electric
1Deceased, March 23, 1971.

timer. Dark incubations were usually carried out in the main

laboratory by wrapping the beakers in two layers of aluminum

foil. Chlorophyll and protochlorophyll were extracted and determined as described earlier (2, 5).
RESULTS
Chlorophyll Accumulation by Cotyledons with Hook Attached. Our first objective was to determine whether the lag
phase in cotyledons excised with and without hypocotyl hooks
could be removed by a short light pretreatment followed by a
dark incubation. Four-day-old etiolated cucumber cotyledon
pairs were excised with and without hypocotyl hooks and exposed to white fluorescent light, 240 ft-c, at 28 C for 5 min.
They were wrapped in aluminum foil to exclude light and incubated at 28 C for 5 hr and 55 min. Controls were excised
in the same way but wrapped immediately in aluminum foil
without light pretreatment and incubated in the dark for 6 hr.

After the dark incubation the aluminum foil was removed

from all samples and the cotyledons were exposed to white
light for 4 hr. The 5-min light pretreatment had no effect on
the hookless cotyledons, which accumulated as little chlorophyll as the nonpretreated hookless cotyledons (Table I); however, it had a marked effect on cotyledons excised with hooks
(Table I). The chlorophyll accumulated (93.6 ,ug of total
chlorophyll per g fresh wt) under these conditions corresponds
within the experimental error to the amount found after 6 hr
of continuous light (2), indicating that 5 min of light followed
by 5 hr, 55 min of darkness are sufficient to overcome the
short lag phase in cotyledons excised with hooks but have no
effect on the prolonged lag phase of hookless cotyledons.
The lengths of the illumination and of the dark incubation
periods were varied, one at the time, to determine the illumination period necessary to remove the lag phase of etiolated
cucumber cotyledons. Under our experimental conditions
(temperature, light intensity, and light quality) 45 sec of light
followed by 4 hr of darkness proved sufficient to remove the
lag phase in etiolated cotyledons excised with hypocotyl hooks

(Table II).
We previously reported (2) that, when cotyledons with hooks

are exposed to continuous light, maximal chlorophyll accumulation takes place if the hooks are shielded, suggesting that the
photoreceptor is in the cotyledons and that the hook-borne
stimulation is somehow light-sensitive. However, in those experiments the tissue was exposed to white light continuously for
5 hr. One could therefore object that, during such a long period,
enough reflected light might pass below the aluminum foil to
trigger a photoreceptor system in the hypocotyl hook. The experiment was repeated, but this time the wrapping was applied
only during the 45-sec light pretreatment. The aluminum foil
was held in place around the shielded organ with modeling
clay, thus forming a light-tight seal. After the 45-sec light pretreatment the aluminum foil was removed, and all samples
705
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Plant Physiol. Vol. 47, 1971

HARDY, CASTELFRANCO, AND REBEIZ

706

Table 1. Effect of Light Pretreatment oi Chlorophyll

Accumlulationi in Cotyledo,is Excised wit/i and
without Hypocotyl Hook
The tissues were exposed to 240 ft-c of white fluorescenit light
for 5 min, dark-incubated for 5 hr and 55 min, then exposed to
240 ft-c of white light for 4 hr. All treatments were done at 28 C.
Chlorophyll a

b Accumulated
in Cotyledons

Plant Material
Light-

Not lightpretreated

pietreated

pg/gfresh wt

Cotyledons with hypocotyl hooks

Cotyledons without hypocotyl hooks

69.2

93.6
8.9

8.2

were incubated in the dark for 4 hr and then exposed to continuous white light for 2 hr. The results (Table III) confirm
that maximal chlorophyll accumulation occurs when the cotyledons are uncovered and the hooks covered (2).
In order to find out whether the hypocotyl hook exerts a
continued influence on chlorophyll accumulation in the cotyledons after tho removal of the lag phase, five samples of 4-dayold etiolated cucumber coty,ledons were excised with full hooks
and were exposed to 4 hr of continuous white light. The hooks
of the first sample were removed before the beginning of the
illumination, the hooks of the second sample were removed
after 1 hr, those of the third sample after 2 hr, those of the
forth sample after 3 hr; the last sample was incubated with
hooks for the full 4-hr period of illumination. The results
indicate that, once the lag period is over, the hooks are

I~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

Table II. Lag Phase Renioval ini Cuicuniber Cotyledons Excised with
Hypocotyl Hooks, by Variouts Light Pretreatniient a,id
Dark Incubation Contditions
Cotyledons excised with their hypocotyl hooks were exposed
to 240 ft-c of white fluorescent light for the indicated pretreatment
period, then dark-incubated for varying lengths of time, and finally exposed to 240 ft-c of white light for 2 hr. All treatments
were at 28 C.

Table IV. Conitinued Iniflutenice of Hypocotyl Hook oii Chlorophyll

Accuzmuilatio,i ini Coty.ledons
Cotyledons were excised with their hypocotyl hooks and exposed to 240 ft-c of continuous white fluorescent light for various
lengths of time. At the times indicated, the hooks were removed.
Altogether the incubation lasted 4 hr and was carried out at 28 C.
Incubation Conditions

Chlorophyll
a + b Accumulated
in Cotyledons

I ug/g fresh wi
Chlorophyll a + b Accumulated in Cotyledons

Experiment 1. Light
pretreatment
300 sec
0

20.7
7.8

33.1
13.8

Dark incubation, hr
1 3
4
pg/g fresh wt
40.2
17.5

34.4
18.7

1 hr, with hooks

3 hr, without hooks

6.7

33.9
13.9

31.2
16.3

2 hr, with hooks

2 hr, without hooks

14 8
14.

120

300

3 hr, with hooks

1 hr, without hooks

30.3

45.4

42.0

4 hr, with hooks

0 hr, without hooks

85.2

Dark incubation, hr
3
4 1

pg/g fresh wI
Experiment 3. Light
pretreatment
45 sec
0

20.5
14.2

21.2
14.1

21.5
16.3

46.4
21.3

39.1
21.5

45.2
24.5

Table III. Effect of Hypocotyl Hook Shieldinig during 45-sec Light

Pretreatment upon Chlorophyll Accumulationi
Cotyledons excised with hypocotyl hooks had either the hook,
the cotyledon, or neither shielded during the 45-sec exposure to
240 ft-c of white fluorescent light. The tissues were then darkincubated for 4 hr and exposed to 240 ft-c of white light for 2 hr.
All treatments were done at 28 C.
Treatment

Chlorophyll

a + b Accuniulated
in Cotyledons

Cotyledons

Table V. Lag Phase Removal in Hookless Cucumber Cotyledonts by

Various Light Pretreatment anid Dark Incubation Conditions
Cotyledons excised without hypocotyl hooks were exposed to
240 ft-c of continuous white light for the period indicated. They
were then incubated in the dark for varying lengths of time and
finally exposed to continuous white light for 2 hr. All treatments
were done at 28 C.
Chlorophyll a + b Accumulated in Cotyledons
Experiment 1. Dark incubation, 6 hr

Experiment 2. Light pre-

Uncovered
Covered
Uncovered

52.5
64.9
35.0

9.4

15.1

43.4

35.6

32.6

40.2

40.6

Dark incubation, hr
2 1 3 1 4

5 1

ug/g fresh wt

wt

Experiment 3. Light pre-

jg/g fresh WI

17.6
fresh

Light pretreatment, hr
2
3
4

treatment, 2 hr

Hooks
pg/g

Uncovered
Uncovered
Covered

7.6

Light pretreatment, sec

10
20
30
60
Experiment 2. Darkpggfehi
fresh
incubation, 3 hr
Pg/g wt
22.2 34.9 39.5 40.7 44.5 48.5
0

0 hr, with hooks

4 hr, without hooks

treatment, 2 hr
12.4

34.8

37.9

39.9

35.2

Dark incubation, hr
0.33 | 0.67
pg/g fresh wt
23.4
19.6
23.8

0.17

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36.8

32.4

1.0
25.3

Plant

Physiol. Vol. 47, 1971

GREENING OF EXCISED COTYLEDONS

still necessary for continued chlorophyll accumulation during

the period of illumination (Table IV). If the hooks are removed during the lag phase (after 1 hr of illumination) the
same low chlorophyll accumulation is observed as in hookless
cotyledons; beyond that the accumulation varies directly with
the time the hooks are left attached (Table IV).
Chlorophyll Accumulation by Hookless Cotyledons. The
lag phase in hookless cotyledons is not removed by a 5-min
light pretreatment followed by dark incubation (Table I) but
requires a much longer exposure to continuous light (2). A
study was therefore undertaken to determine the minimal light
exposure followed by a dark incubation which would remove
the lag phase of hookless cotyledons. Our results (Table V)
indicate that 2 hr of continuous white light followed by a dark
period are sufficient to remove the lag phase in hookless cotyledon tissue. The optimal length of the dark period appears to
be about 60 min; however, even 10 min have a noticeable effect
on chlorophyll accumulation (Table V).
The above-mentioned results suggested that the hooks may
provide a diffusable substance or substances needed in the
greening process. The chemical nature of these substances has
not been investigated beyond the direct testing of the following compounds: 8-aminolevulinic acid, auxin, gibberellin,
abscisic acid, the cytokinin, 6-benzylamino purine, and
ethylene. None of these was able to replace the hooks completely in our assay system. Indeed, when etiolated cotyledons
excised with and without hooks were incubated in 2.0 ml of
25 mm ALA2 under 4 hr of continuous light, the results (Table
VI) indicated that: (a) ALA does not substitute for the hook
effect upon chlorophyll accumulation; (b) in the presence of
ALA a small amount of nonphototransformable protochlorophyll accumulates; (c) at the concentration used, ALA exerts
a moderate inhibition upon chlorophyll accumulation in cotyledons with hooks attached; this inhibition is consistent with
the findings of Steer in barley (8). When hookless etiolated
cotyledons were incubated with 1 and 10 ,ug/g of IAA, GA3,
ABA, and BA under white light for 3 hr, none of these compounds showed any marked effect on chlorophyll accumulation. Ethylene at 4.4 l/liter in air appeared to replace in
part the hypocotyl hook (Table VII). When the incubation
was performed in Warburg vessels with KOH in the center
well, enhanced chlorophyll accumulation was observed, suggesting that the respiratory CO2 produced by the tissue itself may
have an inhibitory effect on chlorophyll accumulation.

DISCUSSION
The experimental results reported in this paper suggest the
presence of a unknown substance (or substances) involved in
chlorophyll accumulation when etiolated tissues are exposed to
light. In intact sedlings, this component is presumably synthesized in the metabolically active, highly meristematic
hypocotyl hook tissue and translocated to the cotyledons. The
latter expand and green up rapidly on exposure to white
light. When the hook tissue is completely removed and the
cotyledons are exposed to light, a particularly long lag phase
is encountered, although eventually greening does occur (2).
This hook factor is not necessarily a single chemical entity;
rather, it could be a complex mixture, like the one found to
control the elongation in cucumber seedling hypocotyl segments (4).
An investigation of the common growth regulators indicated that, in all likelihood, ethylene plays a role. The greening

2Abbreviations: ALA: 8-aminolevulinic acid; BA:

purine.

6-benzylamino

707

Table VI. Effect of Exogenious 3-Aminolevulic Acid oi Chlorophyll

Accumulation in Excised Cotyledons
Cucumber cotyledons excised with and without hypocotyl hooks
were exposed to 240 ft-c of white fluorescent light for 4 hr at 28 C.
The tissue was bathed in either 2 ml of 25 mm ALA or 2 ml of

H20.
Pigment Accumulated in
Cotyledons
Plant Material

Treatment
Proto-

Chlorophyll

chlorophyll

+b

,pg/g fresh wi

Cotyledons with hooks

Cotyledons without hooks

+
_

6.5
0.0

56.6
85.1

5.8
0.5

5.0
7.6

Table VII. Effect of Ethylene ont Chlorophyll Accumuilationl it

Excised Cucumber Cotyledons
Excised cucumber cotyledons were exposed for 4 hr to 240 ft-c
of fluorescent white light at 28 C. The plant material was contained
in 25-ml Erlenmeyer flasks sealed with soft rubber injection caps.
After the plant material was introduced, the flasks were flushed
for 10 min with air and 4.4,ul/liter ethylene in air. Gas inlet and
outlet were provided by means of hypodermic needles that were
removed after flushing.
Chlorophyll a + b Accumulated
Plant Material

in Cotyledons
in_Cotyledons

No ethylene

Ethylene

pg/g fresh wt

Cotyledons with attached hooks

Hookless cotyledons

58.38
16.35

47.76
30.25

process in etiolated cucumber cotyledons seems hindered by

the accumulation of respiratory CO2 in the incubation vessel.
This effect was also reported by Steer (10). In fact, many
known effects of ethylene, on ripening, growth, and abscision
are competitively inhibited by CO2 (3). When the hypocotyl
hook is exposed to light, there seems to be a decrease in the
hook effect (Table III). Antagonism of light toward the production of ethylene has also been found in several higher plant
systems including pea epicotyl and bean hypocotyl (3).
Finally, our plant material was found to release a considerable amount of ethylene into the atmosphere. When the
tissue was incubated in sealed 25-ml Erlenmeyer flasks for 4
hr at 28 C under continuous light, the following ethylene concentrations were accumulated: cotyledons with attached hooks,
0.0251 + 0.0014 ul/liter; cotyledons plus detached hooks,
0.0464 + 0.0019 ,/liter; hookless cotyledons, 0.0073 +
0.0005 ,ul/fit.-r.
Addition of ethylene (Table VII) enhances the chlorophyll
accumulation in the hookless cotyledons. However, the chlorophyll accumulation in cotyledons with attached hooks is consistently higher; therefore, it appears that other factors beside ethylene might be involved, possibly some component
which cannot diffuse readily though the water or air phase, but
which can be easily translocated from hypocotyl hook to
cotyledon in the intact seedling.
Interorgan synergism with respect to chlorophyll accumulation is not limited to cucumber seedlings. We have observed

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708

HARDY, CASTELFRANCO, AND REBEIZ

that etiolated pea primary leaves green up faster if they are

excised with the epicotyl hook tissue than if they are excised
without. Wolf and Price (10) and Sisler and Klein (7) reported
that etiolated bean primary leaves green up more slowly if the
cotyledons are detached from the seedling.
Jeffery pine embryos must acquire certain unknown substances from the megagametophyte in order to produce chlorophyll in the dark (1, 6). However, if the embryos are grown
under continuous white light, the gametophyte is not needed.
Our main objective in this research is to identify the factors
which mediate such interorgan synergism with respect to
chlorophyll accumulation, when dark-grown seedlings are exposed to light.
1.

LITERATURE CITED
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2. HARDY, S. I., P. A. CASTELPFANCO, AND C. A. REBEIZ. 1970. Effect of the

3.
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6.

7.
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9.
10.

Plant Physiol. Vol. 47, 1971

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Copyright 1971 American Society of Plant Biologists. All rights reserved.