Download PROTEIN AND TOTAL FREE AMINO ACIDS IN THE SHOOT TIP

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Indian J. Plant Physiol., Vol. XXVIII No.3, pp. 277-283 (September 1985)
PROTEIN AND TOTAL FREE AMINO ACIDS IN THE SHOOT TIP AND SUBTENDING LEAVES DURING ONTOGENY, FLORAl;­
TRANSITION AND REVERS[ON OF PINEAPPLE PLANT K.N. MADHUSUDANAN AND S. NANDAKUMAR
Department of Botany, Calicut University, Kerala
(Received: June 29, 1984; Revised: September 6, 1985)
SUMMARY
To'al protein and free amino acids were estimated in Ananas comoslls
(L.) Merr. shoot tip at eleven stages of ontogenic development under
natural environment. Protein and free amino acids decreased in the
shoot tip after two months from planting. In the achlorophyllous part
of leaf. protein content decreased but an increase in amino acids Was
noted. In the chlorophyllous region, how)'ver free amino acids decreased
and an increase in protein was recorded: The most significant ch!lDges
dUring the vegetative phase was the rise in free amino acids in 12 months
old shoot tip. In the fully mature plant protein and total free amino
acids decreased in the shoot tip and also in achIoropbyUous leaf part but
increased by about 3 fold in the chlorophyllous region, without any
change in protein concentration. In the fully evoked state of the shoot
apex (when tbe first kinked bract appeared) protein and amino acid
concentration increased. Except at tbe early stage of infiorescence,
protein concentration was lower in the shoot tip than in the leaf parts.
INTRODUCTION
-----..-___
A number of studies have revealed the association of proteins and free
aniino--acids with the development in shoot apex and in subtending leaves during
transition period and in the leaves during the photoperiodic induction (Bernier,
1971; Bernier, Kinet and Sachs, 1981; Evans, 1971; Vince-prue, 1975; Housley
et al., 1979). Little information is available on the phase of the ontogeny of
the shoot apex and the leaves during flowering. Pineapple plant is unique in its
susceptibility to forced flowering at any growth stage (Collins, 1968), indicating
absence of an obligatory juvenile period. The 'reversion' of the inflorescence
apex to the vegetative state (crown formation) is also distinctive in pineapple
plant. An earlier study from this laboratory has revealed changes in carbohy­
drate during growth and floral transition in the pineapple (Madhusudanan and
..
278
IC.N. MADHUSUDANAN AND S. NANDAICUMAIt.
Nandakumar 1983). The present study is an evaluation of the total protein and
total free amino acids in the shoot tip and subtending leaves of AnontU at
selected stages of ontogeny, with particular reference to transition phase of
flowering under natural conditions and the reversion of the inflorescence apex to
the vegetative condition.
MATERIALS AND METHOD
Pineapple plants (Anonas comosus (L.) Merr., Kew cv.) were raised from
hapas, as described by Madhusudanan et of. (1983). Stages for sample collection
(Table 1) and tissues analysed were the same as reported earlier (Madhusudanan
and Nandakumar, (1983).
Growth
Stage
Time of Collection
Characteristics
1.
Maturehapa
propagule
2.
2-months after planting
rooting
3.
4-montns
4.
6-months
s.
8-months
6.
10-months
7.
12-montbs
8.
14-montbs
9.
10.
11.
"
.. ..
.. ..
.. ..
..
.. ..
,~
1
J
I
>­
I
I
rapidly growing
vegetative state
J
,
Adult. apparently
still vegetative
First kinked bract
evocation
Visible doming. clustering
of bristlelike bracts
organogenesis
All florets difi'erentiated;
no anthesis
reversion
Protein and amino acid detenninatiens: Total protem. was determined by
the method of Lowryet of. (1951). with minor modification (Khanna et of.
1969). "Free amino acids were extracted from 2 g tissue with hot 80% (v/v)
ethanol. 'The ethanol~free preparation was loaded on a column of Dowex 50-x8
and eluted with 0.1 N NH. OH. Total amount of amino acid was calculated
by summing up the contents of individual amino acids separated by two-dimens·
ional descending chromatography using n·butanol. acetic acid, water (100:22:50,
v/v) for the first run and phenol and water (4:1, v/v) as solvents for the second
run (Stepka, 1957; Giri, Radhakrishnan and Vaidyanatb,an, 1952).
279
PROTEIN AND AMINO ACID DUltING'DEVBi.oPMEN'i'Ai STAGBS
,The amount of protein and total amino acids were calculated per g dry
weight of tissues and g starch-free dry solids, as reported by Madhusudanan and
Nandakumar (1983). lhe proportion of amino acids in the free condition was
calculated as follows :
percentage free _ free amino acids. in mg X 100
amino acids - free amino acids
(1.1 x protein). in mg
+
RESULTS
The data for protein and total amino acid concentration are represented in
Fig. I and 2.
01
....... 01
E
-'
o 30
0­
'::
"
l Il.l I ~LLl
-:iI:
."2
>­
~.t;
o
c
10
t.
t'
~·
1; il'
:i
,;,
'~
23'56
E
I t .
<:I
I
•
~
9
to
11
Stages of development
Fig. 1. Total free amino acids in the shoot tip and subtendinl leaves of
A.nQnos comoslU. =: Shoot tip; JIJ achlorophyllous leaf part;
• chlorophyllous leaf part.
300
"
,
.t::Q.o 100
'0
....
"
Q.
8
7
4
5
6
Stages of devE'lopment
3
9
10
b
11
Fig. 2. Total protein in the shoot tip and' subtending leaves of..tfntmtu
com08U4 • .
shoot tip; . DJ achloropbyllous leaf part; • chloro­
phyllous Jeaf .part.
=
280.
:K.:.toI'i~ADHUSUDANAN
'AND S. NANDAIWl4AR,.
Three months old propagule used for planting (stage 1) had comparatively
high concentration of protein and large amino acid pool in its tissues. At stage
2. there was a sharp reduction both in protein alld free amino acids (p<O.OI
each) in the shoot tip. a reduction of protein (P<O.OI) and an increase in free
amino acids (P<O.OI) in the achlorophyllous leaf part. An increase in total
protein (P<O.OI) and an abrupt decrease in the amino aCids pool in the chloro~
phyllous leaf part was also noted.
On transition from stage 7 to stage 8, when the plant was fully mature. if
nodncipientl:y reproductive, there was a decrease in protein and total free amino
acids (P<O.OI each) in the shoot tip. On transition from vegetative (stage 8) to
reproductive state (stage 9). protein and free amino acids increased in the shoot
tip (P<O.Ol each). Increase in protein in shoot tip was not significant during
floral organogenesis (stage 10) and in the early stage of inflorescence (stage II).
IJl
"0
A
20
I
u
I
0
II
0
C
'­
I
E 10
0
,
(]) (!) -
,
\
.\
I
;~
/
I....
~
I \
/
rI
,
Fig 3.
2
3 4 5 6 7 8 9
Stages of development
10' 11
Fig. 3. ~ercentage free a~ino acids
] .
.
, Free amino aeids (total)
[ froteiil X1.1 +fr~~ amilit> acil;ls x 100 In the shoot tIP and
subtending leave' of An,mas co",:osus.
0-0 shoot tip;
0, ~ - '" "!' 0 achlor~phyllous leaf part; .-::-. chlorophy]]ou8 leaf
part.
At all stages, protein concentration in the shoot tip was lower than in the
chlorophylIous)eaf part, wi~ exception of st~ge 11. when the protein concentra~
.tion in the inflorescence was I ~6-fold that of chlorophYllous leaf part. Protein
r
PROTEIN AND AMINO ACID DURING DEVELOPMENTAL STAGES
281
concentration in the chlorophyllous leaf part was nearly the same as the achlo~
rophyllous leaf part in stage 1; at all other stages, the chlorophyllous leaf part
had higher protein concentration.
The concentration of total free amino acids in the shoot tip was more
than in the achlorophyllous leaf part. The free amino acids pool was always
higher in the achlorophyllous than in the chlorophyllous leaf part.
Free amino acids as percentage of total amino acids
The total of free amino acids, percentage, was found highest iti. the shoot
tip at all stages, except at stage 6 (Fig. 3). The percentage of free amino acids
was always found higher in the achloropJiyJIous than in the chlorophyllous leaf
part.
DISCUSSION
Hapa had a high level of carbohydrate in its tissus and this 'was attributed
to its dual source of nutrition, namely, elaboration in its own tissues and
acquisition from the parent plant (Madhusudanan and Nandakumar. 1983). The
observed high level of protein and amino acids in the shoot tip could not be
explained similarly, since the hapa was dependent exclusively on the mother
plant for its mineral nutrition.
The period lapsing before establishing itself in the soil is one of intense
stress for the propagule as a whole. Both protein and free amino acids of the
shoot tip and the protein of the achlorophyllous leaf part appear to be used up
during the first two months after planting of propagule. . It was earlier found
that starch and sugars decreased in stage 2, presumably due to utilization during
the rooting process. The marked reduction in free amino acids concentration
in chlorophyllous leaf part suggested utilization for protein synthesis at the site
or that free amino acids were diverted in increased amounts from the chloro­
phYttous leaf part, for supply to the shoot apex and other sinks.
The forcing of flowering in the pineapple plant by growth factors was
generally carried out at a time when the plant attained 12-14 months growth
(Gifford, 1969; Bartholomew, 1977). The rapidity of the response to applica­
tion of hormone would suggest that internal resources in the plants were being
drawn upon. The 3-fold increrse in the concentration of total free amino acids
at stage 7 (enhanced 3-fold also on starch-free dry weight basis) was without
parallel to any other stage. The reproductive phase appeared to be initiatedl
preceded by a flooding of tissue with free amino acids. The flooding with
282
K.N. MADHUSUDANAN AND S. NANDAKUMAR
sugars was to take place several weeks later, stage 9 (Madhusudanan and
Nandalrumar, 1983).
The dramatic increase in amino acids at the shoot tip in stage 7 was
associated with a 1arge decrease in free amino acids in the leaf parts. It was
presumably during, or a period close to, stage 7 that induction reactions com~
menced in the leaft leading to tbe formation of the flowering principle, for
transmission to the shoot apex.
The increase in protein observed in stage 9, the evoked shoot tip. was in
keeping with the increase in protein content of cells in the apical meristem of
photoperiodic plants (Bernier, 1971) and day-neutral plant (Corson and Gifford.
1969). The increased free amino acid content could be due to the inereased
permeability for solutes, as postulated by Evans (1975). An increased production
of growth factors at the shoot tip would promote the accumulation of meta­
bolites at the shoot tip. The order of incre~e in free amino acids (about 25
per cent) was much lower than that of soluble carbohydrates (6-fold) Madhusu­
danan and Nandakumar. 1983). There was only a small change in the
percentage of total amino acids in the shoot tip during evocation. This contrasted
with the IO-fold increase in the total sugars as a percentage of total metabo­
lizable carbohydrate (Madhusudanan and Nandakumar, 1983).
The higher concentration of protein and amino acids in shoot tip during
stage 10 was understandable since differentiation of floral primordia required
additional synthesis of protein. During reversion, a further synthesis of protein
seemed to be necessary to meet the needs of processes which may involve
dedifferentiation and redifferentiation. That the shoot tip relied heavily on free
amino acids was evident from the higher percentage content of free amino acids
in all stages with an exception (stage 6).
ACKNOWLEDGEMENTS
The authors are grateful to professor P.S. Krishnan, Emeritus professor
in Biochemistry, for his interest in the investigation and to Professor B.K. Nayar,
Head of the Department. for laboratory facilities.
REFERENCES
Bartholomew, D.P. (1977). Inflorescence development of pineapple (AnolUU como.us L.)
Merr.) induced to flower with ethephon.Bot. Goz~. 138: 312-.20.
Bernier. G. (1971). Structural and metabolic changes in the shoot apex'in transition to
flowering. Can. J. Bot., 49: 803-19.
PROTEIN AND AMINO ACID DURING DEVELOPMENTAL STAGES
283
Bernier, G •• Kinet, J.M. and Sachs, R.M. (19SI). The physiology of Flowering. Vol. 1.
C.R.C. Press, Boca Raton, Florida.
Collins, J.L. (1968). The pineapple, Leonard HilI, London.
Corson, G.E. and Gifford, E.M. (1969). Histochemical studies of the shoot apex of Datura
stramonium during transition to flowering. Phytomorphology, 19: 119-96.
Evans, L.T. (1971). Flower induction and the fiotigen concept. Ann. Rev. Plant Physiol.,
365-94.
n:
Evans L.T. (1975). Day length and the Flowering of Plants. W.A. Benjamin, Inc, California.
Gifford, E.M. (1969). Initiation and early development of the inflorescence in pineapple
(Ananas comosus'Smooth. Cayenne') treated with acetylene. Amer. J. Bot., 48 :
657-66.
Girl, K.V., Rl\dhakrishnan, A.N., and Vaidyanathan, C.S. (1952). Quantitative estimation of
amino acids after separation by paper chromatography. Anal. Chem .• 24: 1677-1.
Housley, T.L., Schradar, L.E., Miller, M. and Setter, T.L. (1979). Partitioning of 14C­
photo synthate, and long distance translocation of amino acids in prefiowering and
ftowering, nodulated and nonnodulated soybeans. Plllltt Physiol., 64 : 94-8.
Khanna, S.K., Mattoo, R.L.• Viswanathan, P,N.• Tewari, C.P. and SanwaI. G.G. (1969).
Colorimetric determination of protein and orthophosphate in plant tissues rich in
phenolics. Indian J. Biochem., (;: 21·'.
Lowry, O.H., Rosebrough, N.J., Farr, A.L. and RandaU. R.J. (19'1). Protein measurement
with Folin phenol reagent. J. Bioi. Chem., 193 : 26'-75.
Madhus~anan, K.N.• Nabeesa. E., Umadevi, V. and Nandakumar, S. (1983). Crop growth
pattern and the propagule differentiation of 20 varieties in pineapple. Scientia
Hort;c., 18 : 215-24.
Madhusudanan, K.N. and Nandakumar, S. (1983). Carbohydrate changes in shoot tip and
subtending leaves during ontogenetic development of pineapple. Z. pflanzen­
physiol., 110 : 42~38.
Stepka, W. (1957). Indentification of Amino acids by paper chromatography. Methods
Enzymol., Ill, S04-28.
Vince-prue, D. (1975). Photoperiodism in plants. McGraw-HilI, London.