California State Univer·sity, Northridge
THE ROLE OF PHENOLIC COMPOUNDS
q
IN THE GROWTH OF LIGHT AND DARK GROvJN SEEDLINGS
A thesis submitted in partial satlsfac·tion of the
req~irements
for the degree of Master of Science in
Biology
by
--
Dominick Clayton Salerno
August, 1977
The Thesis of Dominick Clayton Salerno is approved:
Dr.
Wi I I i am Ernboden
-·-y-;~
..
;:...-----:r~·-llo...-~-----"'"----..-
-
----'~-..__~~--
Dr. Mary R(Cor·coran, Committee Chairman
CALIFORNIA STATE UNIVERSITY, NORTHRIDGE
i
. i
'
II
PREFACE
I would like to thank Dr. Harry Highkin and Dr. Wi I I lam Emboden
for serving on my graduate committee.
I also thank Dr. Mary Corcoran for her advice, understanding,
humor and extreme good-natured tolerance of my many examples of unorthodox behavior and actions.
I am most appreciative to Debra Koutnik for advice, technical
assistance, the discussions we've had both scientific and otherwise,
her love and for onl ightenlng me to many facets of I ife and relationships ihat I was heretofore ignorant of.
Special appreciation is to Jean Salerno for her many years of
love, patience, support and encouragement.
iIi
TABLE OF CONTENTS
Preface ......•............
:1
•••••••••••••••••••••••••••••••••
Abstract .........................................
t:
i
.............
it
v
Introduction ................................................ .
Materials and Methods ....•...........................•....... 4
Resu Its . .......... ., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Figure
Total Phenolic Content and Length Measurements of
Light and Dark Grown Dwarf Peas .... ;~ .............. 13
Figure 2
Pathway of Phenolic Biosynthesis via
Phenylalanine ammonia-lyase ........................ 14
Figure 3 Standard Curve of Tannic Acid via
Fo I in-Den is Reagent ......•........................• 15
Table
Total Phenolic Content of Extracts from
Light and Dark Grown Seedlings ...................... 16
Table 2
Total Gal lotannin Content of Extracts from
Light and Dark Grown Seed! ings ..........•........... 18
Table 3
Effect of Tannic Acid on Growth of
E-t- i o Iated Dwarf Peas ................................ 20
Table 4
Effect of Tannic Acid & Dialyzed Pea Extracts
on Let-t-uce Hypocoty I Growth ................•........ 21
Table 5
Effect of Light on Growth of Etiolated Dwarf Peas ... 22
D'iscussion ...... ~ ............•..•.
'a
••••••••••
.,
••••••••••••••
•
23
Literature Cited ....................••..•..•................. 26
iv
ABSTRACT
THE ROLE OF PHENOLIC COMPOUNDS
IN THE GROVJTH OF LIGHT AND DARK GROViN SEEDLINGS
by
Dominick Clayton Salerno
Master of Science In Biology
Seed! ings of five species of plants were grown in darkness or
I ight for ·two wePks.
extracted.
Shoots were measured in length and exhaustively
The total phenolic content was determined colorimetrica! ly
after addition of Fol in-Denis reagent.
In all species, the lengths of
the dark grown seed! ings were longer than the corresponding I ight
grown plants.
It was found that the.endogenous levels of phenolics
were substantially higher in the I ight grown seed I ings.
The gal lo-
tannin content was also found to be higher in light grown seed! ings.
Dwarf pea seed! ings were grown in continuous darkness and watered
with water alone or solutions of tannic acid CO.Sg/1 and S.Og/1 ).
At the lower concentration, there was a stimulatory-effect on growth
relative to the water control.
At higher concentration, although
there \vas an increase in tota I Iength, there was substantia I reduction
v
in the length of the first two internodes and development of a fourth
internode not present in either the water control or the lower concentration of tannic acid.
Lettuce hypocotyl sections were incubated in
I ight or darkness in water or solutions of GAy tannic acid, dialyzed
extracts from I ight or dark grown peas, or mixtures of GA
acid or pea extracts.
3
and tannic
Tannic acid and pea extracts inhibited growth
of hypocotyl sections regardless of I ighting conditions.
The degree
of inhibition was greater in dark incubated sections and extracts
from I ight grown peas were more inhibitory than extracts from dark
grown peas.
These data suggest that phenolics may be involved and
play a regulatory role in the I ight-mediated inhibition of growth.
vi
INTRODUCTION
The morphogenic effects of etiolation upon seedling development
are wei !-documented.
The first publ lshed experimental observations
were by Bonnet (1754), who noted that in peas long internodes and
smal I yellow leaves were produced in darkness and that these plants
became green after exposure to I ight for 24 hours.
MacDougal (1903),
in the most comprehensive monograph to date on the anatomical and
morpho Iog i ca I eHects of et i o Iat ion, docurr1ented the great variation
in many different species, both monocots and d i cots.
/\mong the most
common effects of dark growth in dicotyledonous plants are extensive
internode elongation; retarded leaf development and, in many cases,
formation of a distinct apical hook.
Upon exposure to I ight, numerous morphogenic and physiological
growth responses occur, however, the mechanisms of most of these are
little understood
O~ohr,
1972).
One of the more dramatic effects of
I ight to alter growth patterns, particularly
~rihibiting
growth in
length, has caused growth hormones to be considered as mediating
substances.
Galston & Baker (1951) demonstrated that the concentration of
indole acetic acid (IAA) necessary to el lcit
sections
wa~
seed I ings.
g~owth
in pea epicotyl
affected by prior I ight treatment of the etiolated
Maximum growth occurred In sections from seed I ings grown
~eeded
to induce growth when
the seed! ings had been exposed to red I ight.
They also noted that
in continuous darkness.
More IAA was
IAA would not induce growth in sections from fully I ight grown
2
seed I i ngs.
In another report, Hi II man & Ga I ston ( 1957), noted that
the activity of IAA-oxidase in peas was significantly reduced by red1ight treatment.
These and other reports (Galston, 1969) would seem
to indicate that red-1 ight reduction of IAA-oxidase activity is a
regulatory step in the I ight inhibition of growth.
are inconsistencies In this Interpretation.
However, there
Application of exogenous
IAA wi I I not reverse red-1 ight mediated growth inhibition.
would be expected that since red-i ight
io~e~s
iAA~)xidase
Also, it
act
ivi~les,
there should be a concommitant increase in the level of endogenous
auxins within I ight grovm plants and thus, ! ight grown plants would
be expected to show more growth than etiolated plants.
In contrast, gibberel I ins can reverse I ight inhibition of growth
and restor"e shoot length to that char-acteristic of etiolation (Lockhart, 1965; Lockhart, 1959; Lockhart & Gottschal I, 1959).
Lockhart
suggested that I ight probably acted on gibberel I ins by reducing the
synthesis or possibly by destroying gibberel i ins.
In eithel- case
dark grown plants would be expected to contain more glbberel lin than
light grown plants.
The vJor-k of Jones & Lang (1968) and Kende 8, Lang
(1964) wiTh dwarf and normal peas demonstraTed that there is no
quantitative difference In difference in endogenous gibberel I In levels
between I i ght and dark grov"n peas.
f<ende and Lang ( 1964) cone I uded
that I ight treatment resulted in a decreased sensitivity to gibbera I I ins.
Their· suggest Ion was supported by Musgrave, et a I . , ( I 969)
who showed that I ight reduced the growth responsiveness of tissues to
glbberel I ins and also reduced the binding of
GA
5
to normally responsive tissues.
radioac~fvely
label led
3
The difference in GA sensitivity between I ight and dark grown
plants could be explained by the action of growth inhibitors.
A
I ight induced increase in inhibitor could reduce the amount of growth
caused by an unchanged amount of GA.
Because of its potent growth
inhibitory properties, the abscisic acid content of I ight and dark
grown peas was assayed by Kende & Kays (1971), but no differences in
levels were found.
Tannins and certain sma I I phenol i cs have been repor-ted to be
GA antagonists (Corcoran et al ., 1972 and Green & Corcoran, 1975).
They inhibit only the growth induced by GA, not the endogenous
growth, and increasing the amount of GA completely reversed inhibition.
At saturating doses of GA, the plants treated with the promoter
alone v1ere indistinguishable from those treated with promoter and
tannin.
The reversal suggests that tannins are involved In a
gibberel I In mechanism.
If tannins or, more generally, phenolics are involved In the
regulation of gibberel I in induced growth In the plant, the
a~ount
of
phenolics wouid be expected to show an inverse correlation with
growth.
In the light-dark system, light gr·own piElnts would be
expected to have a higher phenolic content than etiolated plants.
In this study, the total phenolic and gal lotannin content was
determined in I ight and dark grown seed! ings of five species.
Information is presented suggesting that tannins and other phenol lcs
may be involved in the mediation of I ight inhibited growth and
possibly explain the difference in gibberel I in sensitivity between
i ight and dark grown seed! ings.
MATERIALS AND METHODS
PLANT MATERIALS
Growth
Seeds of peas, Pisum sativum L. cvs. Alaska, a tal I strain, and
Progress #9, a dwarf strain, were planted In a I: I mixture of sol I
and vermicul lte and allowed to grow for 14 days in a growth chamber
(Conviron Model E7) at 20+.05 Cor in a greenhouse at 24+4 C.
Seed! ings In the growth chamber were grown either in a 16 hour photoperiod with I lght furnished by a bank of eight 40 watt Sylvania
Life! ine fluorescent tubes and four 30 watt Ken-Rad incandescent
bulbs, or in continuous darkness provided by a I ightproof wooden box.
In the greenhouse, seed! lngs were grown in an 18 hour photoperiod
from natural day! ight supplemented with fluorescent I ighting or In
continuous darkness In a dark box.
Shoots were harvested, measured
in length from the first node to the apical meristem, counted, weighed
and frozen at -10 C.
Seeds of radish,
Raphanu~
sativus L. cv. Cherry Bel I, and
cucumber, Cucumis sativus L. cv. National Plckl lng, were planted In
sand and grown for 14 days at 25±10 C In the greenhouse.
Seedlings
were kept In continuous darkness or in an 18 hour photoperiod as
described above.
measured from
th~
Shoots were harvested as above.
Radishes were
point of emergence of the uppermost branch to the
apex of the longest cotyledon; cucumbers were measured from the same
upper root area to the juncture of the cotyledons.
Seeds of lettuce, Lactuca sativa L. cv. Arctic King, were
4
5
planted in vermiculite and grown for seven days at 24 ± .05 C in the
growth chamber, either In continuous darkness or in a 16 hour photoperiod.
The plants were harvested and treated as above.
Shoot length
was measured from the point of emergence of the uppermost branch root
to the tip of the cotyledons.
Extraction
Frozen shoots were homogenized in acetone (400 ml. If fresh
weight was 50 g., 150 mi. if 10-15 g. F\11) in a Sorval 0mni-m1xer.
The homogenate was placed in a eel lulose extraction thimble and
extraction conducted in a Soxhlet extractor under a nitrogen atmosphere for 2 1/2 hours.
The solvent was changed to acetone:water in
proportions such that the ratio in the extraction chamber was ! :I.
Nitrogen was reintroduced and extraction repeated for 2 1/2 hours.
The solvent was then changed to water and extraction .continued as
before.
The three extracts were combined and stored under nitrogen in
a refrigerator at 6 C.
The combined extracts were then evaporated
under vacuum at 40 C to a final concentration equivalent to the
extract from I g. FW/1 ml. of final solution.
The concentrated
extracts were used for phenolic determination or were dialyzed for use
In the lettuce hypocotyl bioassay.
Extracts of dwarf and tal I peas grown in I ight or darkness were
dialyzed in one I iter of water at 6 C for 24 hours.
dialyzed five more times in a simi lar'manner.
The samples were
The six I lters of
dialysate were combined and evaporated under vacuum at 40 C to give
a final con6entration equivalent to the extract from I g. FW/ml.
6
PHENOLIC DETERMINATION
Tota I Pheno I i cs
Total phenolic content was determined calorimetrically after
adding Fol in-Denis reagent CA. 0. A. C.).
Pre! iminary tests showed
no difference in phenolic content between dialyzed and non-dialyzed
extract, so extracts without dialysis were used in these tests.
AI iquots of the concentrated extracts were diluted I :9 in water and
a I ml. a! iquot of the dilution was
adde~
to 75 ml. of water.
To
this was added 5 ml. of Fol in-Den is reagent and the contents mixed
wei!.
Ten ml. of saturated sodium carbonate solution was added,
stirred, and water .added to bring volume to 100 ml.
The reactants
were allowed to stand for 30 minutes to permit ful I color development
and absorbance was determined at a wavelength of 760 nm in a Bausch
and Lomb Spectronic 20 colorimeter.
The absorbance readings CA
760
)
were compared to a standard curve using tannic acid (Baker & Adamson
reagent grade) as a reference.
The phenolics were expressed as
mi I I I grams of tannic acid equivalents.
Gallotannins
Gal lotannin content was determfned by lead acetate precipitation
(Robinson, 1975; Wallet a!., 1969).
One mi. of concentrated, non-
didlyzed extract was mixed thoroughly with 4 ml. of 10% acetic acid
and 2 ml. of 10% lead acetate was added.
The resulting solution was
shaken for one minute and allowed to stand for ten minutes for ful I
precipitation of
~al
lotannins to occur.
The precipitate was centri-
fuged, collected and suspended in 15 ml. methanol.
An excess of
hydrogen sulfide was bubbled through the suspension to free the
7
gal lotannins and the suspension centrifuged to separate a precipitate
of lead sulfide.
The supernatant was freeze-dried and the dried
residue dissolved in 75 ml. of water.
mined as described above.
Total pheno11cs were deter-
The gal lotannins were expressed as
mi I I igrams of tannic acid equivalents as described above.
PLANT GROWTH TESTS
Dwarf Peas
'owarf peas, cv. Progress #9, were grown for 14 days in
vermiculite at 20 C in continuous darkness.
The plants were watered
with water or tannic acid solutions four times beginning on the fifth
day after plantingi and every other day thereafter.
The concentra-
tions of tannic acid applied were 0.5 g./1. and 5.0 g./1.
On the
14th day, the plants were harvested and measurements of the individual
Internodes and ove ra I I Iength were taken.
Lettuc~
Hypocotyl Bioassay
This bioassay, developed by Silk and Jones (1975) was selected
for relative ease, reproducible results and because the hypocotyls
exhibit etiolation when grown in the dark.
sativ~
Lettuce seeds, Lactuca
L. cv. Arctic King, were sown in petri dishes, germinated for
36 hours in darkness, and the hypocotyls excised according to the
procedure described by Stuart and Jones (1977).
The hypocotyl
sections were measured, as described below and incubated in smal I
plastic petri dishes, containing 4 mL. of test solution, for 56 hours
at 27 C in darkness or continuous I ight in a growth chamber.
I
• j
!
The test solutions consisted of water alone, solutions of
gibberel I lc
~cid
or tannic acid, extracts from dialyzed I ight and
8
dark grown dwarf and tal I peas, and mixtures of GA 3 with tannic acid
or the extracts.
The GA 3 (potassium salt-Calbiochem) was dissolved
in absolute ethanol at a concentration of I ug./ml.
Four ml. of this
solution were applied to the appropriate culture dish and the ethanol
was allowed to evaporate.
The GA 3 was left behind and 4 ml. of water,
tannic acid solution or pea extract was added.
concentration of GA
3
was 4 ug./ml.
Thus, the final
Tannic acid solutions were made
up in water at concentrations of 100 ug. and 1000 ug./ml.
The
dialyzed pea extracts were diluted I :4 in water to give the following
approximate amounts of phenolics:
Light grown dwarf seed I i ngs
1200 ug.
Light grown ta II seed I ings
968 ug.
Dark grovm dwarf seed I ings
354 ug.
Dark grown ta II seed I i ngs
322 ug.
Length measurements of the hypocotyl sections were determined
before and after incubation, by the method used by Stuart and Jones.
Dishes were placed on a columnated microscope i I luminator with
overhead I ighting, and the images were focused on paper, magnified
14x.
The images were traced on paper and measured with a map
reader <Keutfel & Esser, Switzerland).
percentage increase in the mean final
Growth is expressed as the
length relative to the mean
initial length for ten hypocotyl sections:
%AL
=
Ln f.1 na I ·- Ln .1 n 1. t.1a I/L n ...
t. I X I00
1 n 1 1a
where n is the number of hypocotyls and L is the length.
RESULTS
Plant Materials and Phenol Jc Content
Five species of plants were grown In either 16 or 18 hour
photoperiods or in contlnous darkness.
They were measured and
extracted to quantitatively assay the total phenolic and gal lotannin
content.
Shoot lengths of alI etiolated plants were consistently
taller than the corresponding I ight grown material (Table I), and
the t6tal phenolic content of dark grown material was significantly
less than that of the I i ght grown seed I i ngs.
In dwarf pea, the
difference in length of dark vs. I ight grown plants was about 300%.
The diffefence in phenolic content of I ight vx. dark grown plants
was 188.1% expressed as mg. total phenolics/gram fresh weight, and
269.3% as total phenolics/plant.
graph (Figure I).
These data are depicted in a bar
A repeat with a second extract from another group
of seed I ings gave similar results (Table I).
Although the tal I vari-
ety of pea exhibited the characteristic overgrowth In the dark, the
percent difference In length was not as striking as in the other
species studied.
The total phenolic content, however, was signifi-
cantly higher in the I ight grown plants on both a mg./g. FW and
mg./plant basis.
In lettuce, there was an extremely high difference
in plenol ics when expressed as mg./plant.
This is due prlmari ly to
the large weight difference between I ight and dark grown seed I lngs.
The trends of greater length in etiolated seed!1ngs and higher
phenolic content in I ight grown seed!
remaining species tested.
9
i~gs
are reflected in the
10
The quantitative determination of gal lotannins (Table 2) shows
that in alI cases but one the amount of gal lotannins per g. FW is
greater in pI ants grown in the I i ght than in the dark.
In two cases
the difference is not great and in one there is no difference.
Although the gal lotannin content is higher in the I ight, the percent
gal lotannin relative to
~he
total phenolic content is lower in the
I ight with only the radish having a higher percent in the I ight.
The fraction of total phenolics as gal lotannins is relatively smal I
in a I I cases.
~t
i ol ated Pea Seed I i ng Test
Dwarf peas, Progress #9, were grown in darkness for 14 days
and watered with water alone or tannic acid solutions.
The
seed I ings
were measured on the 14th day for overal I and individual internode
lengths.
The data (Table 3) show there Is a growth stimulatory effect
on total length and alI internode lengths
rel~tive
control at the lower concentration of tannic acid.
to the water
At the higher
concentration there is a significant reduction in the lengths of the
first and second internodes, 28% and 16%, respectively.
There was
approximately a 60% increase in the length of the third internode and
the total length was greater.
It should also be noted that only in
plants treated with the higher concentration of tannic acid was there
a development of a fourth internode.
Lettuce
~pocotyl
Bioassay
In the second growth test the effects of GA , tannic acid,
3
dialyzed extracts from two varieties of I ight and dark grown peas
II
and mixtures of GA 3 and tannic acid or pea extracts were tested.
Lettuce hypocotyl sections were incubated for 56 hours in the various
test solutions in continuous I ight or darkness.
The data represent
the mean percent change in in it i a I Iength to f ina I Iength of ten
sections.
The percent change in length for the water controls and
GA 3 treatments is greater in the dark than in the light (Table 4).
A number of observations concerning the effects of phenolic compounds
are apparent.
Tannic acid and pea extracts, regardless of I ighting
during growth, inhibit the growth of hypocotyl sections.
The extract
from both I ight grown varieties of peas were more inhibitory than the
corresponding extracts from dark grown varieties, although the
inhibition from the extract of I ight grown, tal I peas is comparable
to that of the extracts of dark grown peas under etiolated conditions.
It can also be seen that there is a greater degree of inhibition of
etiolated hypocotyls by tannic acid and pea extracts than of sections
grown in the I ight.
GA 3 reversed the inhibition by tannic acid and
promoted growth relative to the water controls in both the I ight and
dark treatments.
At a ratio of 1:1000 (GA-tannic acid), there is a
greater inhibition in the dark than in the I ight.
Only extracts from I ight grown peas were tested for GA reversibi I ity.
GA was effective in reversing inhibition in sections
incubated in both light and dark, however, again there is a greater
inhibition in the dark grown sections.
Table· 4 shows that GA reversal
of inhibition caused by tal I pea extract was 6% in the dark and growth
was enhanced in the I ight.
extract by 42% in the dark.
GA reversed the inhibition of dwarf pea
Lack of remaining extract prevented
12
testing of reversibi I ity in the I ight.
13
Figure I.
Total Phenolic Content and Length Measurements of Light
and Dark grown Dwarf Peas.
Seedlings of dwarf pea, Progress #9, were frozen, homogenized and
extracted sequentially with acetone, acetone:water, and water.
Combined extracts were concentrated and treated with Fol in-Denis
reagent.
Absorbancy was determined by spectrophotometer at 760 mu.
Data on phenolics expressed as tannic acid equivalents.
r--
+c
3:
(1J
lL
0)
..........
E
E
E
2.0
250
1.6
200
0)
Length
Total Phenolics
150
..........
0)
E
rmm
l"" t
111 HI
~
--
~-
Ill! II
111111
1.2
0..
1.5
t- I • 2
IIIII!
!IIIII
Iff ff I
0.9
Ill! II
Iliff!
I 1l ff I
111111
0.8
100
IIIII I
!IIIII
I ill !I
Iff 111
111111
0.6
111111
0.4
0
50
n
n::::::
Ill! II
t 11111
t tt 11 t
II !Itt
l" ff t
Till It
mm
rmm
11111!
II lilt
11!1!1
!IIIII
IIIII!
I !till
mg/g FW
r.rnm:
1 1l If I
1111 Iff
!IIIII
f: 11 ff t
0.3
11111!
I lll11
II Till!
imur
rng/plant
0
CatE~ch
ins
Leu~oanthocy5nldins
Phenylalanine
PAL
Cinnamlc acid
p-Coumaric acid
smaller phenolics,
i.e., Caffeic acid
Fe ru I i c ac i d
Flavonoids
Figure 2.
Pathway of Phenol lc Biosynthesis via Phenylalanine ammonia-lyase.
+::-
15
Figure 3.
Standard Curve of Tannic Acid with Fol in-Den is
Reagent.
.
\()
L1\
(f)
-t-
c
Q)
(1J
>
:"J
o-
.
''<j-
Q)
-o
(J
<:(
(J
c
c
(1J
J'(')
f-
rn
E
N
16
Table I.
Total Phenolic Content of Extracts from Light and Dark
Grown Seed I i ngs.
Seed! ing shoots were frozen, homogenized and extracted
sequentially with acetone, acetone:water, and water.
Extracts were
pooled and tested with Fol in-Denis reagent and absorbancy determined
by spectrophotometer at 760 mu.
acid equivaLents.
Data on phenolics given as tannic
Length (mm)
Sample
Pheno 11 cs
(mg/g FW) (mg?PTeint)
Difference
in length
(%)
Pea - AI aska
ll ght
dark
199.0
I. 12
I ~ I0
225.0
0.40
0.48
2.67
0.42
I .63
0.21
3.04
0.49
I .83
0.22
50.16
I .37
0.82
173.23
0.49
0.25
38.7
I. 63
0.75
152. I
0.59
0.24
48.51
I .04
0.68
0.64
0.02
Difference in
_mg/g FW
<%)
Difference ln
mg/plant
<%)
13. I
178.6
129.6
247.0
64.3
105.4
358.8
66.1
I 14.3
245.4
176.6
231.5
293.0
176.3
212.5
91.7
62.2
3300.0
Radish
II ght
dark
I ight
dark
29.05
100.8
26.83
123.1
Cucumber
It ght
dark
light
dark
Lettuce
light
dark
93.1
,...j
18
Table 2.
Total Gal lotannin Content of Extracts from Light and Dark
Grown Seed I ings.
Seedling shoots were frozen, homogenized and extracted sequentially with acetone, acetone:water, and water.
Combined extracts
were concentrated, acidified and treated with lead acetate to
precipitate gal lotannins.
The precipitate was treated with H2S to
release galloi"annins which were then treated with Fol in-Denis reagent
and absorbancy determined by spectrophotometer at 760 mu.
as tannic acid equivalents.
Data given
Sample
Ga! lotannins
(ug/g_ f\'1)
Gallotannins
(%of total phenoI ics from Table I)
Difference in
Gallotannins
(%)
I. 90
II. 76
Pea - Progress #9
dark
28.5
25-.5
5.76
light
95.0
5.59
dark
34.8
7.56
light
28.4
2.54
dark
28.5
7.09
4.69
dark
125.0
,65.0
light
160.0
7.84
78. I
4.27
I i ght
85.0
6.20
dark
40.0
8.08
light
95.0
9.15
dark
85.0
13.28
II ght
171 .43
Pea - A I aska
0
Radish
I ight
dark
4.00
92.31
~
192.68
Cucumber
112.50
Lettuce
II. 76
\.0
Table 3.
Effect of Tannic Acid on Growth of Etiolated Dwarf Peas.
Progress #9 peas, 20 plants per treatment.
Measured 14 days after planting.
Grown in complete dark-
ness in vermiculite with water alone or tannic acid solutions (0.5 g/1 or 5.0 g/1) added through the
roots 4 times starting on the fifth day after planting and every other day thereafter.
AI I measure-
ments in mm.
Treatment
1st
Internode Length
2nd
3rd
4th
Total
Length
Water
60.53 ± 3.18
69. 18 ± 3.30
73.88 ± 6.66
0
203.59
0.5 g/1 tannic acid
64.05 ± 2.56
92.35 ± 3.35
98.20 ± 3.69
0
254.60
5.0 g/1 tannic acid
43.53 ± 2.06
57.90
± 2.62
I I8. 80 ;!; 3. I9
55.95 ± 5.27
276. 18
N
1':)
21
I,
Table 4.
Effect of Tannic Acid
& Dialyzed Pea Extracts on Lettuce
Hypocotyl Growth.
Ten hypocotyl sections in 4ml treatment solution and incubated in
continuous I ight or darkness for 56 hours.
I ug/ml.
GA concentration is
Tannic acid concentrations are IOOug/ml and IOOOug/ml.
Extracts were diluted I :4 in water to give expressed concentrations.
TREATMENT
%LENGTH
--
%OF
CONTROL
%GROWTH
INHIBITED
DARK
WATER
107.89
100.00
0.00
GA
TA 400ug
TA 4000ug
245.42
45.10
14.74
154.02
227.47
41 .80
13.66
0.00
58.20
86.34
142.76
GA-TA I: 1000
LD 1200ug
I I .49
10.65
2.13
I .97
0.00
89.35
98.03
LN 968ug
G/\-LD
20.86
19.33
80.67
46.06
42.69
57.30
GA-LN
72.71
67.39
32.61
DO 354ug
26.87
24.90
75.09
ON 322ug
22.60
20.95
79.05
54.58
100.00
0.00
GA
TA 400ug
209.17
383.24
0.00
34.33
62.90
37.10
TA 4000ug
-8.98
100.00
GA-TA I: I 00
-4.90
70.58
0.00
GA-T A I : I 000
LD 1200ug
33.26
2.35
129.31
60.94
4.31
LN 968ug
27.90
68.44
51. 12
39.06
95.69
48.88
125.39
0.00
40.72
34.33
74.61
62.90
25.39
GA-TA I: I00
LIGHT
WATER
GA-LN
DO 354ug
ON 322ug
37.10
,
Table 5. Effect of Light on Growth of Etiolated Dwarf Peas.
Little Marvel peas, 20 plants per treatment.
Measured 8 days after planting.
Grown In complete
darkness or darkness i I luminated on the fourth day for 10 minutes at 100 em from a 40 watt Mazda lamp
2
covered with a Corning fi Iter No. 348 corresponding to± I erg/cm /sec. AI I measurements fn mm.
(Taken from Went, F. 1941.
Amer. Jour. Bot. 28:33-95, Table 2).
Treatment
1st
2nd
Internode Length
3rd
4th
Total
Length
Darkness
64.5 ± 2.4
52.1 ± I .8
71.6±3.9
0.8
189.0
I I luminated
50.3
42.6 ± 2.6
96.4 ± I .5
8.9
198.2
±
I, 5
N
N
DISCUSSION
There Is a great
dea~
of evidence for the photoinducted
increases of specific phenolic compounds (Bottomley et al ., 1966;
Engelsma, 1965; Mumford et at., 1961, Galston, 1969).
In a number of
instances, this effect can be attributable to the activity of
phenylalanine ammonialyase (PAL) (Attridge & Smith, 1967; Engelsma,
1968).
The PAL enzyme system catalyzes the deamination of
L-phenylalanine tq trans-cinnamic acid, which is the precursor of a
great many phenolic compounds (Figure 2).
The rate of PAL synthesis
has been shown to be control led by phytochrome, which is activated
by I ight at a wavelength of 650 nm. (Mohr, 1972; Zucker, 1972; Smith,
1975).
It is of particular interest to n.ote that Attridge and Smith
(!967) found that the phytochrome-mediated rise in PAL activity has
a time-course almost Identical to the rise in phenolic content in
etiolated peas.
They also noted that light had no effect on shikimate:
i
NADP oxidoreductase activity, another enzyme involved in phenolic bio-.
synthes i.;.
This can exp Ia in the presence of basa I Ieve Is of pheno I i cs ·
in etiolated plants.
The evidence presented in this study demonstrates an inverse
correlation between the endogenous levels of phenolic compounds and
the amount of growth In etiolated and I lght grown seed! ings.
It is
suggested that phenolics may be involved in a regulatory role,
specifically through a gibbers! I In-mechanism,· In the I lght-medtated
Inhibition of growth.
The action spectrum for the I ight inhibition of stem growth has
been determined by Went (1941) and Borthwick et al ., (1951) and was
23
24
found to be maximal at a wavelength of 650nm., which also induces
activity in phytochrome and thus, PAL.
Comparison of data presented
here (Table 3) and data taken from Went (1941) (reproduced in
Table 5) shows a striking paral lei between the effect of tannic acid
and short i I lumination on the growth of etiolated dwarf peas.
In
both investigations, one can see that there was a significant degree
of inhibition in the length of the first two internodes, a stimulation
of the third and fourth internodes, and an increase in overal I stem
length.
It is of especial interest to note that the fourth internode
in the coni·rol was extremely smal I or nonexistent in both of these
investigations.
Smal I phenolic compounds are known to stimulate
growth at low concentrations (Thimann & Bonner, 1949; Nitsch &
Nitsch, 1961 ), and the stimulatory effect of tannic acid at the low
concentration may be similar.
Went concluded that the overgrowth
was due to a compensatory growth mechanism, in which the younger
internode grew more quickly due to the avai labi I ity of certain growth
substances previously monopolized by the I ight inhibited, older
internode.
This conclusion is supported by two different works.
Musgrave et al ., (1960) have shown that radioactively label led GA 5
was accumulated more rapidly in apical tissues than in basal tissues
after I ight treatment and that I ight reduced the binding of GA 5 to
basal tissues.
Neskovic & Sjaus (1974) found an increase in the
amounts of gibberel I in in the upper internodes of etiolated pea
seedlings after treatment_ with red-1 ight.
fie acid
ca~
enter an
lntact~stem
Thus, it is seen that tan-.
and demonstrate an inhibitory
I
effect on endogenous internode growth.
In another study on a
25
different system, Jacobson &Corcoran (1976) demonstrated that
tannins could enter barley seeds and inhibit the synthesis of enzymes
control led by endogenous gibberell ins.
The present study suggests
that these reports can be interpreted such that I ight inhibits growth
by lowering tissue sensitivity through the production of phenolic
compounds that interfere with the binding of gibberel I in to its
specific receptors.
Further support of phenolic Inhibition is seen in the lettuce
hypocotyl bioassay.
Both tannic acid and dialyzed pea extracts were
found to be inhibitory to hypocotyl growth in both the I ight and
darkness.
This inhibition was greater in the etiolated sections,
thus Indicating a greater sensitivity to phenolic compounds, as
would be expected if fewer phenolics were present.
This may also
explain why GA 3 had a lower degree of reversal of inhibition in the
etiolated sections.
The dialyzed extracts from the I ight grown peas
were more inhibitory than the corresponding extracts from dark grown
peas.
This is supported by the findings of Mumford et al. (1961 ),
and Hi I lman & Galston
(~957),
who reported the isolation of a
dialyzable, I ight-induced phenolic Inhibitor of fAA-oxidase from
peas.
They found that extracts from I ight grown peas were more
inhibitory than the extracts from the dark growr1 plants in a pea
section test.
The mechanism of action of phenolic compounds as inhibitors of
growth is unknown.
However, the data· presented here suggest a causal.
relationship between phenolics and gibberel I in in the I ight-mediated
inhibition of growth.
LITERATURE CITED
Attridge,T.H., H. Smith. 1967, Phytochrome mediated increase in the
level of PAL activity in the terminal buds of Pisu~ sativum.
Biochim. Biophys. Acta 148:805
Bonnet, C. l754.
Usage das feu i I Ies. (fide MacOouga I )
Borthwick, H.A. et al. 1951. Action spectrum for inhibition of stem
growth in dark grown seed/ ings of barley. Bot. Gaz. I 13:95
Bottomley, W. et al. 1966.
I I I. Phytochem. 5:117
Co;-coran, M.R. et al.
PI. ~hysiol. 49:323
1972.
Flavonoid complexes in
Pisu~
sativum,
Tannins as glbberell in antagonists.
Engelsma, G. 1965. Photo-induced hydroxylation of cinnamic acid in
gherkin hypocotyls. Na~ure 208:1il7
Enge I sma, G. 1968.
gherkin seed! ings.
Photoinduction of phenylalanine deaminase in
Planta (Berl) 82:355
Galston, A.W. 1969. Flavonoids and photomorphogenesis in peas.
In: J. Harbourne and T. Swain, Perspectives in phytochemistry.
Academic Press, N.Y.
Galston, A.W., R.S. Baker. 1951. Studies on the physiology of
I i ght action, I I I . Am. Jour. Bot. 38: 190
Gr·een,F.B., M.R. Corcoran. 1975. Inhibitory action of five tannins
on growth induced by several gibberel I ins. PI. Physiol. 56:801
Hi llman,W.S., A.W. Galston. 1957. Inductive contrcJi of I.A.A-oxidase
activity by red-1 ight and near infra-red I ight. PI. Physiol. 32:129
Jacobson, A., M.R. Corcoran. 1977. Tannins as gibberel I in antagonists in the synthesis of a-amylase and acid phosphatase by barley
seeds. PI. Physiol. 59:129
Jones,R.L., A. Lang. 1968. Extractable and diffusible gibberel I ins
from light and dark grown pea seed I ings. PI. Physiol. 43:629
Kende,H. ,S. Kays. 1971. The level of (+)-abscisic acid in dvtc.rf
pea shoots. Naturwissenschaften 58:524
A. Lang. 1964: Gibberellins and I ight inhibition of stern
growth in peas. PI. Physiol. 39:435
Kende)~:.,
26
27
Lockhart, J. 1956. Reversal of the 1 ight inhibition pea stem growth
by the gibberel I ins. Proc. Nat. Acad. Sci. 42:841
Lockhart, J. 1959. Studies on the mechanism of stem growth inhibition
by visible radiation. PI. Physiol. 34:457
Lockhart, J. & V. Gottschal I. 1959. Growth response of Alaska pea
seed I ings to visible radiation and GA. PI. Physiol. 34:460
MacDougal, D. T. 1903. The influence of I ight and darkness upon growth
and development. N.Y. Bot. Gard. Mem. Vol. I I
Mohr, H. 1972. Lectures on photomorphogenesis.
Mumford, F. et al. 1961.
PI. Physiol 36:752
Springer-Verlag, N. Y.
An inhibitor of IAA oxidase from pea tips.
Musgrave, A. 1969. In vivo binding of radioactive GA in dwarf pea
shoots. Planta 89:165
Neskovic, M. &1·. Sjaus. 1974. The role of endogenous gibberel I in1ike substances and inhibitors in the growth of pea internodes.
Biologia Plantarum (Praha) 16:57
Nitsch, J.P. & C. Nitsch. 1961. Synergistes naturals des auxines et
des gibberel I ines. Bul I. Soc. Botan. France, 108:349-362
Pro, !vl. 1952. Report on spectrophotometric determination of tannins
in wine and whiskies. A. 0. A. C. 35:255
Robinson, T. 1925. The organic constituents of higher plants.
gess Pub. Co., Minneapolis Minn. p 64
Bur-
Russel, D. W. & A. W. Galston. 1969. Blockage by gibberel I ic acid of
phytochrome effects on growth, auxin responses and flavonoids
synthesis in ·at Io Iated pea internodes. PI . Phys i o I 44: 121 I
Silk, W. K. & R. Jones. 1975. Gibberel I in response in lettuce hypocotyl sections. PI. Physiol. 56:267
Smith, H. 1975. Phytochrome and Photomorphogenesis.
London 235 pages
McGraw Hi I I,
Stuart, D. and R. Jones. 1977. Roles of extensibi I ity & turgor in
GA- and dark stimulated growth. Pl. Physiol. ·59:61
Thimann, K. V. & W. Bonner. 1949. Inhibition of plant growth by protoanemonin & coumarin and its prevention by BAL. Proc. Nat. Acad.
Sci. U. S. 35:272
28
Wal I, M. E. et al. 1969. Plant antitumor agents I I I: A convenient
separation of tannins from other plant constituents. Jour. Pharm.
Sci. 58:839
Went, F. 1941.
28:83.
Zucker, M. 1972.
Effects of I ight on stem and leaf growth.
Enzymes and I ight.
A. J. B.
Ann. Rev. PI. Physiol. 23:133