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Studies on the Comparative Biosynthetic Activities of Embryo and Suspensor in Tropaeolum majus L.
Studies on the Comparative Biosynthetic Activities of Embryo and Suspensor in Tropaeolum majus L. PREM LATA BHALLA,
M. B.
SINGH,
and C. P.
MALIK
Department of Botany, Punjab Agricultural University, Ludhiana-141004, India Received December 16, 1980 . Accepted February 25, 1981
Summary The embryo, suspensor and suspensor haustorium of Tropaeolum maju5 were analysed separately for cellular content, protein content and cellular rates of synthesis of RNA and proteins. The pattern of synthesis differed in suspensor and the haustorium at all stages of embryo development: values for the suspensor were higher than the embryo. The observed data are correlated with the functions of suspensor.
Key words: Tropaeolum maju5, embryo, suspensor, RNA synthesis, protein synthesis.
Introduction Embryogenesis in Tropaeolum maju5 1., garden nasturtium, was described by Walker (1947) and Nagl (1976). Its suspensor is several milimeters long, tripartite embryonic organ which develops precociously and becomes dominant part of the early embryo. The function of suspensor is usually considered to anchor the embryo and to position it in the nutritionally favourable environment. However, recent evidences indicate that suspensor plays a vital role in plant embryogenesis and this organ is compared with the trophoblast of mammals (Nagl, 1973). Studies of Nag! (1976) and Nag! and Kuhner (1976) showed that the cells of embryo and suspensor of Tropaeolum possessed divergent internal structural features. Nagl (1976 b) also repor,ted the presence of giant chromosomes in the suspensor of this species. The nuclear DNA content of cells of embryo proper are only 2C, whereas all the nuclei of suspensor cells are endopolyploid up to 2048C. All these studies indicated that suspensor cells are having biosynthetic capacity higher than the embryo proper. In the present studies, we have examined the biosynthetic activities of the suspensor and embryo proper as regards to the synthesis of ribonucleic acids and proteins. The observed changes are correlated with the functional aspects of the suspensor. Materials and Methods Fresh unripe fruits of different developmental stages of Tropaeolum majus L. were harvested and embryos alongwith suspensors were dissected out with the aid of a dissecting
z. Pflanzenphysiol. Bd.
103. S. 115-119. 1981.
116
PREM
LATA BHALLA, M. B.
SINGH and C. P.
MALIK
microscope. For all the studies embryo, suspensor proper and carpel haustorium were taken as separate units. For the measurement of cell number per embryo or suspensor, the excised embryo-suspensors were transferred to 0.5 ml of 1 0/ 0 pectinase prepared in 0.1 M acetate buffer, pH 5.0. Following incubation for 20 hrs t the cells were separated by shaking the tubes gently on a cyclomixer and the cell number per unit volume was measured using a standard haemocytometer (see Jensen, 1962). Total protein content was determined by the method of Lowry et a1. (1951) and incorporation of amino acids into the proteins was determined by the method of Mans and Novelli (1961) . Rate of RNA synthesis was studied by the method employed by Nakashima (1976) .
Results and Discussion The embryo of Tropaeolum consists of two distinct cell populations. Of .these the suspensor consists of numerically stabilized population of similar cells whereas the embryo proper consists of cell population which enlarges by continued mitosis (Fig. 1) and diversifies through histodifferentiation. Figure 2 shows total protein content of the embryo, the suspensor and the carpel haustorium. The protein content of the embryonic cells was far lesser than the cells of the suspensor and the carpel haustorium. The suspensor of heart embryo had maximum proteins and its level declined rapidly. The protein content in the carpel haustorium was highest in the globular embryo, declined in heart embryo and then it showed a slight increase in the early cotyledonary stage. On the whole, cells of the suspensor and its modificaEmbryo agQ (days)
7
Ii
10
E ]1.00
~
.." ~
~
0/
:,0
•
•,
1200
800
~
E e
'"•
"
400
/ 0I'·" , 120xl0
2000
~ 1600
~ ~
14
L:
G
H
3
;: e
3600 3200
~
2400
~
~
~
~
0
0
1600
800
0
C
Fig. 1: Cell numbers of embryo (. . ), suspensor (0--0) and carpel haustorium (. . ) at globular (G), heamhaped (H) and cotyledonary (C) stage, of development. Z. Pflanzenphysiol. Bd. 103. S. 115- 119. 1981.
Biosynthetic activities of embryo and suspensor in Tropaeolum
117
Embryo a9_ (days)
7
ro
"
10 r'--r-------r---------~__,35 0
315
IB 0
'"e• "
~
-• " Do
11
~
0
10
35 G
H
Stage
c
.Fig. 2: Cell ular content of protein in the emb ryo (e---e ), suspensor (0----0) and carpel haustori um at three Stages of emb ryo development.
<. ---. )
tion (i.e. carpel haustorium) had protein content always higher than the embryo cells of the comparable developmental stages. Sussex et al. (1973) reported that in Phaseolus coccineus the suspensor cellular protein le vel was lowest in the earliest stage and increased gradually throu ghout its development. However, data from Tropa eolum are quite different. It may be added that structurally Tropa eolum suspensor is more complex since it possesses modifications like the placental and the carpel haustoria. The cellular rate of protein synthesis is expressed as incorporation of radioactively labelled amino acids per cell. In the embryo the cellular rate of incorporation into the protein was highest in the ea rly stage and remained constant up to the earl y cotyledonary stage (Fig. 3). Later stages we re not analysed. Presumably, in the mature embryo there is an increase in the protein synthesising capacity of the cotyledona ry cells - a fac t indicated by the accumulation of storage proteins dur ing seed maturation. In cells of the suspensor the rate of incorporation was invariably higher than in the cells of embryo proper and ca rpel haustorium. In fact, in the suspenso r the cellular rate of incorporation was maximal in the heart-shaped stage. Similar trend was noticed in the cells of the ca rpel hautor ium. The rate of RNA synthesis is expressed as incorporation of radioactively labelled uridine into RNA per cell (Fig. 4). The rate of RNA synthesis was highest in all the three parts of the globular embryo. Whereas in the embryo the rate of RNA sy nth esis per cell was always low, the cells of the suspensor or the carpel haustorium of the same stage showed high RNA synthesis. In embryo, on the other hand, the rate of RNA synthesis per cell was highest in the early stages and declined thereafter. Z. Pjlanzenphy,iol. Bd. 103. S. 115- 119. 1981.
118
M. B.
PREM LATA BHALLA,
SINGH
and C. P.
MALIK
Embryoqg. (days)
• ~" ~
3 81'10
If
J
"2
I .~
:v
g
o ·5 E o
~o
10
7
~
e _ e Embryo
Susp.nsor _ Haustorium
0_0
_
61'10
/.~
'1' 10
J
21'10
"
--.
J
•
• ____ 0
.
•
•
c
H
G
Fig. 3: Cellular rates of uC-amino acid incorporation into protein in the embryo (. suspensor (0--0 ) and carpel haustorium (. - -. ) during development.
. ),
Embryo a9' (days) 7
10
"
~~-----,------~-,
21'10'
HJxlO'
,"'1'10' ] ' ·21'10' 11'10'
8xlO'
•
6 1'10'
~ ~
,~
J -
41'10' 21'10' G
H
c
Fig. 4: Cellular rates of 3H-uridine incorporation into RNA in embryo (••----4.1), suspensor ( 0 -0 ) and carpel haustorium (1.1 --1.1) durin g three stages of development.
With the progressive development, suspensor cells accumulated more protein and synthesized RNA and proteins at higher rate than the cells of embryo at the corresponding stage. The suspensor and its haustoria have cells fewer than the organogenetic part of the embryo. At all the stages examined, especially during early development of embryo, cellular protein content in suspensor exceeded that of the
z. Pflanzenphy,iol. Bd.
103. S. 115-119. 1981.
Biosynthetic activities of embryo and suspensor in Tropa eolum
119
organogenetic part. This is particularly eVti dent during the early developmental stages when the biosynthetic activity in the suspensor cells might be impor.tant for the embryo development. Evidently, suspensor plays a vital role in regulating the early development of the organogenetic part of the embryo. I t may be added that embryo-suspensor system of Tropaeolum is much more elaborate and complex as compa red to the simple suspensor of Phaseolus species. The morphological diversification of th e suspensor into the two haustoria giving rise to three distinct parts may have di stinct functional implications for their functional diversification. From the above studies it is also apparent that the carpel haustorial cells possibly play a dominant role in absorption and transport of metabolites whereas suspensor proper cells were concerned with act ive biosynthesis as well as transport. Present investigations thus ,throw abundant light on the divergent biosynchetic potentialities of embryo suspensor and its different morphological mod ifications i.e. haustoria. Acknowledgements This work was financed by CSIR, New Delhi, and forms a part of the Ph . D. thesis submitted to Pun jab Agricultural University, Ludhiana, by P .L.B.
References J ENSEN, W. A.: Botanical histochemistry - P rinciples and Practices. W. H . Freeman and Co. San Francisco and London, 1962. LOWRY, O. H., N. J. ROSEBROUGH, A. L. FARR, and R. J. RANDALL: Protein measurement with the Folin phenol reagent. J. BioL Chern. 193, 265- 275 (1951). MANS, R . J. and G. D. NOVEL LI: Measurement of incorporation of radioactive amino acids into protein by a filter -paper disc method. Arch. Biochem. Biophys. 94, 48- 53 (1961 ). NAGL, W.: The angiosperm suspensor and the mammali an trophoblast: organs with simila r cell st ructu re and function. BulL Soc. Bot. France. Mem. 289- 302 (1973). Early embryogenesis in Tropaeolum majus L. Ultr astructure of the embryo suspensor. Biochem. Phy, iol. Pflanzen. 170,253-260 ( 1976). - Ea rl y embryogenesis in TropaeoLum majus L. Evolution of nuclear DNA content and polyteny in the suspensor. Plant Sci. Lett . 7, 1-8 (1976 b). NAGL, W. and S. KOHNER: Early embryogenesis in Tropaeolum majus: diversificat ion of plastids. Plama 133, 15- 19 (1976). NAKASHIMA, H.: Diurnal rhythm of uridine inco rporation into RNA regulated by twO lightperceiving systems in a longday duckweed, Lemna gibba G3. Plant and Cell Physiol. 17,206-217 (1976). SUSSEX, I. , M. CLUTTER, V. WALBOT, and T. BRADY: Biosynthetic activity of the suspensor of Phaseo/us coccineus. Caryologia 25, 261 -272 (1973). WALKER, T. T.: Megasporogenesis and embryo development in Tropaeolum majus L. Bull. Torrey Bot. Club. 74, 240-249 (1947).
z. Pi/anzenphy,io/. Od.
103. S. 115-119. 1981.
M. B.
SINGH,
and C. P.
MALIK
Department of Botany, Punjab Agricultural University, Ludhiana-141004, India Received December 16, 1980 . Accepted February 25, 1981
Summary The embryo, suspensor and suspensor haustorium of Tropaeolum maju5 were analysed separately for cellular content, protein content and cellular rates of synthesis of RNA and proteins. The pattern of synthesis differed in suspensor and the haustorium at all stages of embryo development: values for the suspensor were higher than the embryo. The observed data are correlated with the functions of suspensor.
Key words: Tropaeolum maju5, embryo, suspensor, RNA synthesis, protein synthesis.
Introduction Embryogenesis in Tropaeolum maju5 1., garden nasturtium, was described by Walker (1947) and Nagl (1976). Its suspensor is several milimeters long, tripartite embryonic organ which develops precociously and becomes dominant part of the early embryo. The function of suspensor is usually considered to anchor the embryo and to position it in the nutritionally favourable environment. However, recent evidences indicate that suspensor plays a vital role in plant embryogenesis and this organ is compared with the trophoblast of mammals (Nagl, 1973). Studies of Nag! (1976) and Nag! and Kuhner (1976) showed that the cells of embryo and suspensor of Tropaeolum possessed divergent internal structural features. Nagl (1976 b) also repor,ted the presence of giant chromosomes in the suspensor of this species. The nuclear DNA content of cells of embryo proper are only 2C, whereas all the nuclei of suspensor cells are endopolyploid up to 2048C. All these studies indicated that suspensor cells are having biosynthetic capacity higher than the embryo proper. In the present studies, we have examined the biosynthetic activities of the suspensor and embryo proper as regards to the synthesis of ribonucleic acids and proteins. The observed changes are correlated with the functional aspects of the suspensor. Materials and Methods Fresh unripe fruits of different developmental stages of Tropaeolum majus L. were harvested and embryos alongwith suspensors were dissected out with the aid of a dissecting
z. Pflanzenphysiol. Bd.
103. S. 115-119. 1981.
116
PREM
LATA BHALLA, M. B.
SINGH and C. P.
MALIK
microscope. For all the studies embryo, suspensor proper and carpel haustorium were taken as separate units. For the measurement of cell number per embryo or suspensor, the excised embryo-suspensors were transferred to 0.5 ml of 1 0/ 0 pectinase prepared in 0.1 M acetate buffer, pH 5.0. Following incubation for 20 hrs t the cells were separated by shaking the tubes gently on a cyclomixer and the cell number per unit volume was measured using a standard haemocytometer (see Jensen, 1962). Total protein content was determined by the method of Lowry et a1. (1951) and incorporation of amino acids into the proteins was determined by the method of Mans and Novelli (1961) . Rate of RNA synthesis was studied by the method employed by Nakashima (1976) .
Results and Discussion The embryo of Tropaeolum consists of two distinct cell populations. Of .these the suspensor consists of numerically stabilized population of similar cells whereas the embryo proper consists of cell population which enlarges by continued mitosis (Fig. 1) and diversifies through histodifferentiation. Figure 2 shows total protein content of the embryo, the suspensor and the carpel haustorium. The protein content of the embryonic cells was far lesser than the cells of the suspensor and the carpel haustorium. The suspensor of heart embryo had maximum proteins and its level declined rapidly. The protein content in the carpel haustorium was highest in the globular embryo, declined in heart embryo and then it showed a slight increase in the early cotyledonary stage. On the whole, cells of the suspensor and its modificaEmbryo agQ (days)
7
Ii
10
E ]1.00
~
.." ~
~
0/
:,0
•
•,
1200
800
~
E e
'"•
"
400
/ 0I'·" , 120xl0
2000
~ 1600
~ ~
14
L:
G
H
3
;: e
3600 3200
~
2400
~
~
~
~
0
0
1600
800
0
C
Fig. 1: Cell numbers of embryo (. . ), suspensor (0--0) and carpel haustorium (. . ) at globular (G), heamhaped (H) and cotyledonary (C) stage, of development. Z. Pflanzenphysiol. Bd. 103. S. 115- 119. 1981.
Biosynthetic activities of embryo and suspensor in Tropaeolum
117
Embryo a9_ (days)
7
ro
"
10 r'--r-------r---------~__,35 0
315
IB 0
'"e• "
~
-• " Do
11
~
0
10
35 G
H
Stage
c
.Fig. 2: Cell ular content of protein in the emb ryo (e---e ), suspensor (0----0) and carpel haustori um at three Stages of emb ryo development.
<. ---. )
tion (i.e. carpel haustorium) had protein content always higher than the embryo cells of the comparable developmental stages. Sussex et al. (1973) reported that in Phaseolus coccineus the suspensor cellular protein le vel was lowest in the earliest stage and increased gradually throu ghout its development. However, data from Tropa eolum are quite different. It may be added that structurally Tropa eolum suspensor is more complex since it possesses modifications like the placental and the carpel haustoria. The cellular rate of protein synthesis is expressed as incorporation of radioactively labelled amino acids per cell. In the embryo the cellular rate of incorporation into the protein was highest in the ea rly stage and remained constant up to the earl y cotyledonary stage (Fig. 3). Later stages we re not analysed. Presumably, in the mature embryo there is an increase in the protein synthesising capacity of the cotyledona ry cells - a fac t indicated by the accumulation of storage proteins dur ing seed maturation. In cells of the suspensor the rate of incorporation was invariably higher than in the cells of embryo proper and ca rpel haustorium. In fact, in the suspenso r the cellular rate of incorporation was maximal in the heart-shaped stage. Similar trend was noticed in the cells of the ca rpel hautor ium. The rate of RNA synthesis is expressed as incorporation of radioactively labelled uridine into RNA per cell (Fig. 4). The rate of RNA synthesis was highest in all the three parts of the globular embryo. Whereas in the embryo the rate of RNA sy nth esis per cell was always low, the cells of the suspensor or the carpel haustorium of the same stage showed high RNA synthesis. In embryo, on the other hand, the rate of RNA synthesis per cell was highest in the early stages and declined thereafter. Z. Pjlanzenphy,iol. Bd. 103. S. 115- 119. 1981.
118
M. B.
PREM LATA BHALLA,
SINGH
and C. P.
MALIK
Embryoqg. (days)
• ~" ~
3 81'10
If
J
"2
I .~
:v
g
o ·5 E o
~o
10
7
~
e _ e Embryo
Susp.nsor _ Haustorium
0_0
_
61'10
/.~
'1' 10
J
21'10
"
--.
J
•
• ____ 0
.
•
•
c
H
G
Fig. 3: Cellular rates of uC-amino acid incorporation into protein in the embryo (. suspensor (0--0 ) and carpel haustorium (. - -. ) during development.
. ),
Embryo a9' (days) 7
10
"
~~-----,------~-,
21'10'
HJxlO'
,"'1'10' ] ' ·21'10' 11'10'
8xlO'
•
6 1'10'
~ ~
,~
J -
41'10' 21'10' G
H
c
Fig. 4: Cellular rates of 3H-uridine incorporation into RNA in embryo (••----4.1), suspensor ( 0 -0 ) and carpel haustorium (1.1 --1.1) durin g three stages of development.
With the progressive development, suspensor cells accumulated more protein and synthesized RNA and proteins at higher rate than the cells of embryo at the corresponding stage. The suspensor and its haustoria have cells fewer than the organogenetic part of the embryo. At all the stages examined, especially during early development of embryo, cellular protein content in suspensor exceeded that of the
z. Pflanzenphy,iol. Bd.
103. S. 115-119. 1981.
Biosynthetic activities of embryo and suspensor in Tropa eolum
119
organogenetic part. This is particularly eVti dent during the early developmental stages when the biosynthetic activity in the suspensor cells might be impor.tant for the embryo development. Evidently, suspensor plays a vital role in regulating the early development of the organogenetic part of the embryo. I t may be added that embryo-suspensor system of Tropaeolum is much more elaborate and complex as compa red to the simple suspensor of Phaseolus species. The morphological diversification of th e suspensor into the two haustoria giving rise to three distinct parts may have di stinct functional implications for their functional diversification. From the above studies it is also apparent that the carpel haustorial cells possibly play a dominant role in absorption and transport of metabolites whereas suspensor proper cells were concerned with act ive biosynthesis as well as transport. Present investigations thus ,throw abundant light on the divergent biosynchetic potentialities of embryo suspensor and its different morphological mod ifications i.e. haustoria. Acknowledgements This work was financed by CSIR, New Delhi, and forms a part of the Ph . D. thesis submitted to Pun jab Agricultural University, Ludhiana, by P .L.B.
References J ENSEN, W. A.: Botanical histochemistry - P rinciples and Practices. W. H . Freeman and Co. San Francisco and London, 1962. LOWRY, O. H., N. J. ROSEBROUGH, A. L. FARR, and R. J. RANDALL: Protein measurement with the Folin phenol reagent. J. BioL Chern. 193, 265- 275 (1951). MANS, R . J. and G. D. NOVEL LI: Measurement of incorporation of radioactive amino acids into protein by a filter -paper disc method. Arch. Biochem. Biophys. 94, 48- 53 (1961 ). NAGL, W.: The angiosperm suspensor and the mammali an trophoblast: organs with simila r cell st ructu re and function. BulL Soc. Bot. France. Mem. 289- 302 (1973). Early embryogenesis in Tropaeolum majus L. Ultr astructure of the embryo suspensor. Biochem. Phy, iol. Pflanzen. 170,253-260 ( 1976). - Ea rl y embryogenesis in TropaeoLum majus L. Evolution of nuclear DNA content and polyteny in the suspensor. Plant Sci. Lett . 7, 1-8 (1976 b). NAGL, W. and S. KOHNER: Early embryogenesis in Tropaeolum majus: diversificat ion of plastids. Plama 133, 15- 19 (1976). NAKASHIMA, H.: Diurnal rhythm of uridine inco rporation into RNA regulated by twO lightperceiving systems in a longday duckweed, Lemna gibba G3. Plant and Cell Physiol. 17,206-217 (1976). SUSSEX, I. , M. CLUTTER, V. WALBOT, and T. BRADY: Biosynthetic activity of the suspensor of Phaseo/us coccineus. Caryologia 25, 261 -272 (1973). WALKER, T. T.: Megasporogenesis and embryo development in Tropaeolum majus L. Bull. Torrey Bot. Club. 74, 240-249 (1947).
z. Pi/anzenphy,io/. Od.
103. S. 115-119. 1981.