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Changes in the Individual Free Amino Acid Concentrations in the Petals of Carnations during the Vase Life of the Flowers
Institute for Botanical Research, University of Potchefstroom, Potchefstroom, Republic of South Africa
Changes in the Individual Free Amino Acid Concentrations in the Petals of Carnations during the Vase Life of the Flowers 1) A.
J. VAN DER WESTHUIZEN and G. H. DE SWARDT2)
With 3 figures Received July 11, 1977· Accepted August 30,1977
Summary Changes in the concentrations of individual free amino acids of petal tissue of carnations (Dianthus caryophyllus L. cv. "White Sim») were studied. In addition, the respiration rate and moisture content of petals were determined as indicators of metabolic activity and degree of senescence. The concentrations of glutamine, glycine, histidine, methionine, phenylalanine, proline, serine, threonine and valine increased throughout the vase life of the flowers. The concentrations of arginine, aspartic acid, isoleucine, leucine, lysine and tyrosine increased initially but decreased thereafter. There was a noticeable decline in the concentration of a-alanine, y-aminobutyric acid and asparagine immediately prior to the climacteric maximum of respiration, whereas during all other times an increase was evident. The concentration of all other free amino acids fluctuated. During the early stages of the vase life of the flowers the increase in free amino acid content is probably due to a de novo synthesis. Proteolysis may contribute significantly to the accumulation of free amino acids during the later stages of the vase life. The decrease in concentration of certain free amino acids towards the end of the vase life may be ascribed to their assimilation as respiration substrates and/or their incorporation during the synthesis of degrading enzymes. The observed accumulation of free ammonia may give rise to an accumulation of asparagine and glutamine with a resultant decrease in aspartic and glutamic acids. Wilting could stimulate the synthesis of (l-alanine and y-aminobutyric acid, whilst inhibiting the synthesis of others such as aspartic acid. Inhibition of amino acid synthesis and the loss of cell integrity may also play an important role in the observed changes in free amino acid concentration. Key words: ageing. senescence, amino acids. Dianthus caryophyllus.
Introduction
During the vase life of carnations in tap water the concentration of total free amino acids, with very few exceptions, tended to increase. Immediately before, or 1) Forms part of aM. Sc. thesis submitted to the Rand Afrikaans University. 2) Department of Botany, Rand Afrikaans University, Johannesburg, Republic of South Africa.
Z. Pflanzenphysiol. Bd. 86. S. 125-134. 1978.
126
A.
J. VAN DER WESTHUIZEN and G. H.
DE SWARDT
during the climacteric maximum for respiration, slight increases or decreases in concentration were observed (VAN DER WESTHUIZEN, 1976). BRUSZEWSKI (1967) suggested that some of the amino acids in the petal tissue of carnations increased or decreased in concentration as the flowers aged. However, no quantitative measurements of individual amino acids were made. Experiments carried out with cut «Better Times» roses during ageing indicated that, with some exceptions, free amino acids and amides accumulated during early stages of senescence, followd. by a decline. Towards the end of the experiment there was an increase in free ammonia which coincided with the respiratory loss of certain free amino acids. These and other results might suggest that, after utilization of a preferential respiratory substrate such as glucose, petal tissues first utilized glumatic acid, and later valine, the leucine, tyrosine, alanine and glutamine (WEINSTEIN,
1957). Initially there may be a de novo synthesis of amino acids in ageing petal tissues of carnations, followed by proteolysis. At the end of the vase life of carnations the free amino acids may serve as respiratory substrates (GREWAR, 1973). A number of workers concluded that increased proteolysis contributed to the accumulation of free amino acids in ageing plant tissues (WEINSTEIN, 1957; THOMPSON et al., 1966; KEMBLE and MACPHERSON, 1954). On the other hand SPENCER and TITUS (1973) suggested that the accumulation of amino acids in senescing apple leaves is due to the de novo synthesis of amino acids. The aim of this study was to acquire more exact information on the changes in the individual free amino acid concentration in the petals during the vase life of cut carnations. This may contribute to a better understanding of senescence of cut carnations. Materials and Methods Fresh carnation flowers (Dianthus caryophyllus L. cv. «White Sim») were acquired from a local nursery. The pedicles were cut at a length of 30 em and the lowest pair of leaves was removed. The flowers were kept in jars containing tap water and were placed in an environmental chamber. The temperature varied between 19 and 22 DC while the relative humidity was approximately 60 %. The day and night lengths were 13 and 11 hours respectively. The light source consisted of twelve 2.35 m tubes (General Electric - F96 PG17 cw) and six 150 w tungsten bulbs. The flowers were placed 1.5 m below the light source where the light intensity was 230J lux. Forty carnations which were distributed at random in the environmental chamber were reserved as controls. Progressive stages of senescence, as judged by comparison with the controls, were sampled by selecting enough flowers from the remainder. The petals of these flowers were removed and bulked before extraction. The method of NQUYEN and PAQUIN (1971) for the extraction and purification of the amino acids was used. The amino acid analysis was done with an automatic Technicon (Model TSM-l) amino acid analyzer. The moisture content of the petals at sampling was determined by drying in an oven at 80 DC for 48 hours. Respiration rates of petals at 20 DC were determined by means of a Gilson respirometer. Z. PJlanzenphysiol. Bd. 86. S. 125-134. 1978.
Free amino acids in the petals of carnations
127
To obtain a common curve for the five replicated values of a specific amino acid the following procedure was followed: The degree of senescence was expressed by using the day on which the climacteric maximum for respiration occurred as reference point. This was done, as the flowers had a longer vase life in some experiments than in others. To establish the amino acid concentrations of the different senescence experiments on a comparable level the following formula was used for the transformation of the data of each experiment:
IV
90
40 90
V
II
'-' ~
I]J
w
~
0
u I]J ~
;:l
'-'
.,.,
0
;:;::
\
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40 90
III
VI
40~--~----r---~---.----.---~~---r---'r---'---~----r---~
4
6
8
10
12
2
4
6
8
10
12
Days after harvest (I, II, III, IV, V, VI = Different ageing experiments) Fig. 1: Changes in the percentage moisture content of petals during the vase life of carnations.
z. PJlanzenphysiol. Rd. 86. S. 125-134. 1978.
128
A.
J. VAN DER WESTHUIZEN and
G. H.
DE SWARDT
Yp = (xp - x) where Y p
=
+
k
transformed value of the amino acid concentration on the day (p) of sampling.
xp
=
x
5 = 1/5 2' xp P=1
average value of the amino acid concentration on day p
k = 1 - lowest value of (xp - x) for all the senescence experiments. 800 IV Q)
::J
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Days after harvest (I, II, III, IV, V, VI = Different ageing experiments Fig. 2: Changes in respiratory rate of petal tissue during the vase life of carnations. Z. P/lanzenphysiol. Bd. 86. S. 125-134. 1978.
Free amino acids in the petals of carnations
129
The constant (k) was introduced to change all the negative values into positive values. For each amino acid the median of the Y I' values of all ageing experiments was plotted, as well as the mean deviation of the median of the Y 1, values.
Results The moisture content decreased drastically after the eighth day after harvest (Fig. 1). 8 """'A""SC=P"7 A-=R=TI""C"-'-A-=C""1D , . - - - - - - - - .
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•
Z. P/lanzenphysiol. Bd. 86. S. 125-134. 1978.
130
A.
J. VAN DER WESTHUIZEN and G. H. DE SWARDT
The climacteric maximum for respiration occurred on the eighth or ninth day after harvest (Fig. 2). The concentration of the following amino acids always increased throughout the vase life: glutamine, glycine, histidine, methionine, phenylalanine, proline, serine, threonine and valine1). Immediately before the climacteric maXimum, the 3 ,--------------------,
3..,.--------------".----------,
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LEUCINE
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PHENYLALANINE
METHIONINE
lYROSINE
o 3
Y-AMINOBUlYRIC ACID
VALINE
6
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2'
6
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2
KM
2
,
Days before and after the climacteric maxi= mum for respiration (KM) Fig. 3 (Continues) 1) The peak for valine on the chromatograms occasionally indicated more than one amino
acid.
z. Pjlanzenphysiol. Bd. 86. S. 125-134. 1978.
Free amino acids in the petals of carnations
131
ORNITHINE
LYSINE
HISTIDINE
6
4
2
KM
2
2
4
2
KM
2•
Days before and after the climacteric maxi= mum for respiration (KM) Fig 3. (Continues) Fig. 3: Changes in the individual free amino acid concentrations of petals during the vase life of carnations.
concentrations of a-alanine, y-aminobutyric acid and asparagine declined, whereas increases were evident at all other times. Concentrations of arginine, aspartic acid, isoleucine, leucine, lysine and tyrosine increased initially but decreased thereafter. The concentrations of all other free amino acids fluctuated (Fig. 3).
Discussion During the early stages of the vase life of carnations the concentration of all the amino acids, except ornithine, increased. This is probably mainly due to a de novo synthesis. This was also suggested by THOMPSON et al. (1966) and SPENCER and TITUS (1973) for senescing plant tissue and BARNETT and NAYLOR (1966) for plants during water stress. V AN DER WESTHUIZEN (1976) found a decrease in protease activity and GREWAR (1973) an increase in total soluble protein content at this stage. Thus proteolysis could probably not contribute significantly to the free amino acid pool during the early stages of the vase life of carnations. The concentration of a number of amino acids continued to increase during the vase life of the flowers. This may be due to several factors. According to BARNETT and NAYLOR (1966) and SPENCER and TITUS (1973) the accumulation of amino acids in senescing plants and plants grown under conditions of water stress is mainly due to Z. Pflanzenphysiol. Ed. 86. S. 125-134. 1978.
132
A.
J. VAN DER WESTHUIZEN
and G. H.
DE SWARDT
a de novo synthesis. WEINSTEIN (1957), THOMPSON et a!. (1966) and KEMBLE and MACPHERSON (1954) however, believe that proteolysis is mainly responsible for the accumulation of the free amino acids during senescence and wilting. The accumulation of some of the free amino acids during the later stages of vase life is thus probably due to proteolysis as well as synthesis. BARASH et a!. (1974) reported that assimilation of ammonia led to an increase in the synthesis of glutamine and asparagine in excised oat leaves. According to CHEN et a!. (1964) and MOTHES (1956) there is an increase in the amide content of plants grown under conditions of water stress. MOTHES (1958) considered glutamine a nitrogen storage compound and an ammonia detoxifier. Visual observations of chromatograms of the free amino acids indicated that the free ammonia content increased during the vase life of the flowers. The moisture content of carnation petal tissue also decreased at a considerable rate. The accumulation of glutamine and asparagine could thus be expected. Factors that could cause proline accumulation are wilting (WALDREN et a!., 1974), increase in free ammonia and decrease in osmotically active compounds such as sugars (SCHOBERT, 1974). BRUSZEWSKI (1967) reported a decrease in carbohydrate concentration in petal tissue during the ageing of carnations. As soon as wilting commenced there was also a marked increase in proline concentration. Methionine was absent or present in trace amounts during the very early stages of the vase life of the flowers. This may suggest that under normal conditions methionine is not important in the metabolic processes. Methionine is generally accepted as a precursor of ethylene (LIEBERMAN et a!., 1965; SAKAI and IMASEKI, 1972). The increase in methionine concentration is probably associated with the increase in ethylene production as found by NICHOLS (1966). It is obvious that the concentration of some of the amino acids increased during the vase life of the flowers whereas the concentration of others decreased after a certain period. Free amino acids may serve as respiration substrate during the later stages of the vase life when the carbohydrate content of the tissue is reduced. GREWAR (1973) and BLACKMAN (1953) came to a similar conclusion. Some of these amino acids, such as aspartic acid are common respiratory substrates (RAMAKRISHNA et aI., 1973). According to BRUSZEWSKI (1967) the carbohydrate concentration decreased noticeably during the later stages of the vase life of carnations. When the oxaloacetic acid concentration is lowered by catabolism during the Krebs cycle, and this cannot be fully compensated for by synthesis from carbohydrate reserves, this may result in reduced aspartic acid levels. Prote!n synthesis may, however, contribute to the reduction in concentration levels of certain amino acids. The synthesis of degrading enzymes can be expected during the later stages of ageing as found by ZAUBERMANN and SCHIFFMANN-NADEL (1972), HOBSON (1963) and HALL (1964) in ripening fruit. ATKIN and SRIVASTAVA (1970) reported a continuous protein synthesis despite a decreased protein content. Proteolysis and protein synthesis are likely concurrent processes.
Z. Pf1anzenphysiol. Bd. 86. S. 125-134. 1978.
Free amino acids in the petals of carnations
133
The observed increase in the ammonia concentration may lead to a decrease in the aspartic and glutamic acid concentration as found by BARASH et al. (1974) during the assimilation of ammonia by excised oat leaves. Wilting and the consequent decrease in oxygen availibility in tissues could stimulate synthesis of a-alanine and ),-aminobutyric acid, whilst inhibiting the synthesis of other amino acids such as aspartic acid (THOMPSON et al., 1966). Arginine may serve as an important nitrogen storage compound in carnation petals as found by HILL-COTTINGHAM and LLOYD-JONES (1973) in certain fruit trees. Certain amino acids, expecially those with common metabolic precursors, may be synthesized at the expense of each other. Feedback inhibition, as well as inhibition by other amino acids, may play an important role in the observed decrease in free amino acids. The loss of cell integrity during the later stages of vase life may drastically influence the relative concentration of protein and amino acids.
It can be concluded that certain amino acids accumulate during vase life mainly as result of nett synthesis and proteolysis. On the other hand a decrease in the concentration of other amino acids, especially near the end of vase life, is apparent. This is probably due to their utilization as respiratory substrates and their role in the synthesis of degrading enzymes.
References ATKIN, R. K. and B. I. S. SRIVASTAVA: Studies on protein synthesis by senescing and kinetin-treated barley leaves. Physiol. Plant. 23, 304-315 (1970). BARASH, I., T. SADON, and H. MOR: Relationship of glutamate dehydrogenase levels to free amino acids, amides and ammonia in excised oat leaves. Plant Cell Physiol. 15, 563-566 (1974). BARNETT, N. M. and A. W. NAYLOR: Amino acid and protein metabolism in Bermuda grass during water stress. Plant Physiol. 41, 1222-1230 (1966). BLACKMAN, F. F.: Respiratory drifts. In: James, W. O. (Ed.): Plant respiration, 40-81, Oxford at the Clarendon Press, London, 1953. BRUSZEWSKI, T. E.: The influence of chemical preservatives on postharvest respiration rates and biochemical changes in carnations (Dianthus caryophyllus L.). Ph. D.-thesis. The Pennsylvania State University, Biochemistry, 1967. CHEN, D., B. KESSLER, and S. P. MONSELISE: Studies on water regime and nitrogen metabolism of Citrus seedlings grown under water stress. Plant Physiol. 39, 379-386 (1964). GREWAR, L.: Biochemiese vcranderinge van sekere verbindings in kroonblare van verouderende angcliere. M. Sc.-thesis Rand Afrikaanse University, Johannesburg, 1973. HALL, C. B.: Cellulase activity in tomato fruits according to portion and maturity. Bot. Gaz. 125,156-157 (1964). HILL-COTTINGHAM, D. G. and C. P. LLOYD-JONES: Metabolism of carbon-14 labelled arginine, citrulline and ornithine in intact apple stems. Physiol. Plant. 29, 125-128 (1973). HOBSON, G. E.: Pectinesterase in normal and abnormal tomato fruit. Biochem. J. 86, 358-365 (1963). KEMBLE, A. R. and H. T. MACPHERSON: Liberation of amino acids in perennial rye grass during wilting. Biochem. J 58, 46-49 (1954). LIEIlERMAN, M., A. T. KUNISHI, and D. A. WARDALE: Ethylene production from methionine. Biochem. J. 97, 449-459 (1965). Z. Pjlanzenphysiol. Bd. 86. S. 125-134. 1978.
134
A. J. VAN DER WESTHUIZEN and G. H. DE SWARDT
MOTHES, K.: Der EinfluB des Wasserzustandes auf Fermentprozesse und Stoffumsatz. In: Ruhland, W. (Ed.), Encyclopedia of plant physiology 3, 656-664, Springer-Verlag, Berlin, 1956. - Ammoniakentgiftung und Aminogruppenvorrat. In: Ruhland, W. (Ed.), Encyclopedia of plant physiology 8,716-762, Springer-Verlag, Berlin, 1958. NGUYEN, S. T. and R. PAQUIN: Methodes d'extraction et de purification des acides amines libres et des proteines de tissus vegetaux. J. Chromatogr. 61, 349-351 (1971). NICHOLS, R.: Ethylene production during senescence of flowers. J. hortic. Sci. 41, 279-290 (1966). RAMAKRISHNA, M., D. B. WANKHEDE, and M. R. RAGHAVENDRA RAO: Incorporation of U- 14 C aspartate into lysine, threonine and other amino acids in bengal gram(Cicer arietinum). Indian J. biochem. biophys. 10, 285-286 (1973). SAKAI, S. and H. IMASEKI: Ethylene biosynthesis: Methionine as an in vivo precursor of ethylene in auxin-treated mungbean hypocotyl segments. Planta (Berlin) 105, 165-173 (1972). SCHOBERT, B.: The influence of water stress on the metabolism of diatoms.!' Osmotic resistance and proline accumulation in Cyclotella meneghiniana, Z. Pflanzenphysiol. 74, 106-120 (1974). SPENCER, P. W. and J. S. TITUS: Apple leaf senescence: Leaf disc compared to attached leaf. Plant Physiol. 51, 89-92 (1973). THOMPSON, J. F., C. R. STEWART, and C. J. MORRIS: Changes in amino acid content of excised leaves during incubation. 1. The effect of water content of leaves and atmospheric oxygen level. Plant Physiol. 41, 1578-1584 (1966). VAN DER WESTHUIZEN, A. J.: Veranderinge in vrye en gebonde aminosuurkonsentrasies in verouderende angelierkroonblare. M.sc.-thesis, Rand Afrikaans University, Johannesburg, 1976. WALDREN, R. P., 1. D. TEARE, and S. W. EHLER: Changes in free proline concentration in sorghum and soybean plants under field conditions. Crop. Sci. 14, 447-450 (1974). WEINSTEIN, L. H.: Senescence of roses.!. Chemical changes associated with senescence of cut «Better Times» roses. Contrib. Boyce Thompson Inst. Plant Res. 19, 33-48 (1957). ZUABERMAN, G. and M. SCHIFFMANN-NADEL: Pectin methylesterase and polygalacturonase in avocado fruit at various stages of development. Plant Physiol. 49, 864-865 (1972).
Mr. A. J. VAN DER WESTHUIZEN, Institute for Botanical Research, University of Pot chefstroom, Potchefstroom, Republic of South Africa.
Z. PJlanzenphysiol. Bd. 86. S. 125-134. 1978.
Changes in the Individual Free Amino Acid Concentrations in the Petals of Carnations during the Vase Life of the Flowers 1) A.
J. VAN DER WESTHUIZEN and G. H. DE SWARDT2)
With 3 figures Received July 11, 1977· Accepted August 30,1977
Summary Changes in the concentrations of individual free amino acids of petal tissue of carnations (Dianthus caryophyllus L. cv. "White Sim») were studied. In addition, the respiration rate and moisture content of petals were determined as indicators of metabolic activity and degree of senescence. The concentrations of glutamine, glycine, histidine, methionine, phenylalanine, proline, serine, threonine and valine increased throughout the vase life of the flowers. The concentrations of arginine, aspartic acid, isoleucine, leucine, lysine and tyrosine increased initially but decreased thereafter. There was a noticeable decline in the concentration of a-alanine, y-aminobutyric acid and asparagine immediately prior to the climacteric maximum of respiration, whereas during all other times an increase was evident. The concentration of all other free amino acids fluctuated. During the early stages of the vase life of the flowers the increase in free amino acid content is probably due to a de novo synthesis. Proteolysis may contribute significantly to the accumulation of free amino acids during the later stages of the vase life. The decrease in concentration of certain free amino acids towards the end of the vase life may be ascribed to their assimilation as respiration substrates and/or their incorporation during the synthesis of degrading enzymes. The observed accumulation of free ammonia may give rise to an accumulation of asparagine and glutamine with a resultant decrease in aspartic and glutamic acids. Wilting could stimulate the synthesis of (l-alanine and y-aminobutyric acid, whilst inhibiting the synthesis of others such as aspartic acid. Inhibition of amino acid synthesis and the loss of cell integrity may also play an important role in the observed changes in free amino acid concentration. Key words: ageing. senescence, amino acids. Dianthus caryophyllus.
Introduction
During the vase life of carnations in tap water the concentration of total free amino acids, with very few exceptions, tended to increase. Immediately before, or 1) Forms part of aM. Sc. thesis submitted to the Rand Afrikaans University. 2) Department of Botany, Rand Afrikaans University, Johannesburg, Republic of South Africa.
Z. Pflanzenphysiol. Bd. 86. S. 125-134. 1978.
126
A.
J. VAN DER WESTHUIZEN and G. H.
DE SWARDT
during the climacteric maximum for respiration, slight increases or decreases in concentration were observed (VAN DER WESTHUIZEN, 1976). BRUSZEWSKI (1967) suggested that some of the amino acids in the petal tissue of carnations increased or decreased in concentration as the flowers aged. However, no quantitative measurements of individual amino acids were made. Experiments carried out with cut «Better Times» roses during ageing indicated that, with some exceptions, free amino acids and amides accumulated during early stages of senescence, followd. by a decline. Towards the end of the experiment there was an increase in free ammonia which coincided with the respiratory loss of certain free amino acids. These and other results might suggest that, after utilization of a preferential respiratory substrate such as glucose, petal tissues first utilized glumatic acid, and later valine, the leucine, tyrosine, alanine and glutamine (WEINSTEIN,
1957). Initially there may be a de novo synthesis of amino acids in ageing petal tissues of carnations, followed by proteolysis. At the end of the vase life of carnations the free amino acids may serve as respiratory substrates (GREWAR, 1973). A number of workers concluded that increased proteolysis contributed to the accumulation of free amino acids in ageing plant tissues (WEINSTEIN, 1957; THOMPSON et al., 1966; KEMBLE and MACPHERSON, 1954). On the other hand SPENCER and TITUS (1973) suggested that the accumulation of amino acids in senescing apple leaves is due to the de novo synthesis of amino acids. The aim of this study was to acquire more exact information on the changes in the individual free amino acid concentration in the petals during the vase life of cut carnations. This may contribute to a better understanding of senescence of cut carnations. Materials and Methods Fresh carnation flowers (Dianthus caryophyllus L. cv. «White Sim») were acquired from a local nursery. The pedicles were cut at a length of 30 em and the lowest pair of leaves was removed. The flowers were kept in jars containing tap water and were placed in an environmental chamber. The temperature varied between 19 and 22 DC while the relative humidity was approximately 60 %. The day and night lengths were 13 and 11 hours respectively. The light source consisted of twelve 2.35 m tubes (General Electric - F96 PG17 cw) and six 150 w tungsten bulbs. The flowers were placed 1.5 m below the light source where the light intensity was 230J lux. Forty carnations which were distributed at random in the environmental chamber were reserved as controls. Progressive stages of senescence, as judged by comparison with the controls, were sampled by selecting enough flowers from the remainder. The petals of these flowers were removed and bulked before extraction. The method of NQUYEN and PAQUIN (1971) for the extraction and purification of the amino acids was used. The amino acid analysis was done with an automatic Technicon (Model TSM-l) amino acid analyzer. The moisture content of the petals at sampling was determined by drying in an oven at 80 DC for 48 hours. Respiration rates of petals at 20 DC were determined by means of a Gilson respirometer. Z. PJlanzenphysiol. Bd. 86. S. 125-134. 1978.
Free amino acids in the petals of carnations
127
To obtain a common curve for the five replicated values of a specific amino acid the following procedure was followed: The degree of senescence was expressed by using the day on which the climacteric maximum for respiration occurred as reference point. This was done, as the flowers had a longer vase life in some experiments than in others. To establish the amino acid concentrations of the different senescence experiments on a comparable level the following formula was used for the transformation of the data of each experiment:
IV
90
40 90
V
II
'-' ~
I]J
w
~
0
u I]J ~
;:l
'-'
.,.,
0
;:;::
\
N
40 90
III
VI
40~--~----r---~---.----.---~~---r---'r---'---~----r---~
4
6
8
10
12
2
4
6
8
10
12
Days after harvest (I, II, III, IV, V, VI = Different ageing experiments) Fig. 1: Changes in the percentage moisture content of petals during the vase life of carnations.
z. PJlanzenphysiol. Rd. 86. S. 125-134. 1978.
128
A.
J. VAN DER WESTHUIZEN and
G. H.
DE SWARDT
Yp = (xp - x) where Y p
=
+
k
transformed value of the amino acid concentration on the day (p) of sampling.
xp
=
x
5 = 1/5 2' xp P=1
average value of the amino acid concentration on day p
k = 1 - lowest value of (xp - x) for all the senescence experiments. 800 IV Q)
::J
rJ) rJ)
.,.,
.w
.-<
<1l
.w Q)
Po
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~
"0 ~ 0 ...... 800 rJ)
II
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Q)
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~
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.,., ~
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rJ)
Q)
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4
6
8
10
12
2
4
6
8
10
12
Days after harvest (I, II, III, IV, V, VI = Different ageing experiments Fig. 2: Changes in respiratory rate of petal tissue during the vase life of carnations. Z. P/lanzenphysiol. Bd. 86. S. 125-134. 1978.
Free amino acids in the petals of carnations
129
The constant (k) was introduced to change all the negative values into positive values. For each amino acid the median of the Y I' values of all ageing experiments was plotted, as well as the mean deviation of the median of the Y 1, values.
Results The moisture content decreased drastically after the eighth day after harvest (Fig. 1). 8 """'A""SC=P"7 A-=R=TI""C"-'-A-=C""1D , . - - - - - - - - .
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6
4
2
KM
2'
6
4
2
KM
2
Days before and after the climacteric maxi= mum for respiration (KM) (6, 4, 2 = days before and 2 = days after) (I = mean deviation)
•
Z. P/lanzenphysiol. Bd. 86. S. 125-134. 1978.
130
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J. VAN DER WESTHUIZEN and G. H. DE SWARDT
The climacteric maximum for respiration occurred on the eighth or ninth day after harvest (Fig. 2). The concentration of the following amino acids always increased throughout the vase life: glutamine, glycine, histidine, methionine, phenylalanine, proline, serine, threonine and valine1). Immediately before the climacteric maXimum, the 3 ,--------------------,
3..,.--------------".----------,
3
LEUCINE
2
5
PHENYLALANINE
METHIONINE
lYROSINE
o 3
Y-AMINOBUlYRIC ACID
VALINE
6
4
2
KM
2'
6
4
2
KM
2
,
Days before and after the climacteric maxi= mum for respiration (KM) Fig. 3 (Continues) 1) The peak for valine on the chromatograms occasionally indicated more than one amino
acid.
z. Pjlanzenphysiol. Bd. 86. S. 125-134. 1978.
Free amino acids in the petals of carnations
131
ORNITHINE
LYSINE
HISTIDINE
6
4
2
KM
2
2
4
2
KM
2•
Days before and after the climacteric maxi= mum for respiration (KM) Fig 3. (Continues) Fig. 3: Changes in the individual free amino acid concentrations of petals during the vase life of carnations.
concentrations of a-alanine, y-aminobutyric acid and asparagine declined, whereas increases were evident at all other times. Concentrations of arginine, aspartic acid, isoleucine, leucine, lysine and tyrosine increased initially but decreased thereafter. The concentrations of all other free amino acids fluctuated (Fig. 3).
Discussion During the early stages of the vase life of carnations the concentration of all the amino acids, except ornithine, increased. This is probably mainly due to a de novo synthesis. This was also suggested by THOMPSON et al. (1966) and SPENCER and TITUS (1973) for senescing plant tissue and BARNETT and NAYLOR (1966) for plants during water stress. V AN DER WESTHUIZEN (1976) found a decrease in protease activity and GREWAR (1973) an increase in total soluble protein content at this stage. Thus proteolysis could probably not contribute significantly to the free amino acid pool during the early stages of the vase life of carnations. The concentration of a number of amino acids continued to increase during the vase life of the flowers. This may be due to several factors. According to BARNETT and NAYLOR (1966) and SPENCER and TITUS (1973) the accumulation of amino acids in senescing plants and plants grown under conditions of water stress is mainly due to Z. Pflanzenphysiol. Ed. 86. S. 125-134. 1978.
132
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and G. H.
DE SWARDT
a de novo synthesis. WEINSTEIN (1957), THOMPSON et a!. (1966) and KEMBLE and MACPHERSON (1954) however, believe that proteolysis is mainly responsible for the accumulation of the free amino acids during senescence and wilting. The accumulation of some of the free amino acids during the later stages of vase life is thus probably due to proteolysis as well as synthesis. BARASH et a!. (1974) reported that assimilation of ammonia led to an increase in the synthesis of glutamine and asparagine in excised oat leaves. According to CHEN et a!. (1964) and MOTHES (1956) there is an increase in the amide content of plants grown under conditions of water stress. MOTHES (1958) considered glutamine a nitrogen storage compound and an ammonia detoxifier. Visual observations of chromatograms of the free amino acids indicated that the free ammonia content increased during the vase life of the flowers. The moisture content of carnation petal tissue also decreased at a considerable rate. The accumulation of glutamine and asparagine could thus be expected. Factors that could cause proline accumulation are wilting (WALDREN et a!., 1974), increase in free ammonia and decrease in osmotically active compounds such as sugars (SCHOBERT, 1974). BRUSZEWSKI (1967) reported a decrease in carbohydrate concentration in petal tissue during the ageing of carnations. As soon as wilting commenced there was also a marked increase in proline concentration. Methionine was absent or present in trace amounts during the very early stages of the vase life of the flowers. This may suggest that under normal conditions methionine is not important in the metabolic processes. Methionine is generally accepted as a precursor of ethylene (LIEBERMAN et a!., 1965; SAKAI and IMASEKI, 1972). The increase in methionine concentration is probably associated with the increase in ethylene production as found by NICHOLS (1966). It is obvious that the concentration of some of the amino acids increased during the vase life of the flowers whereas the concentration of others decreased after a certain period. Free amino acids may serve as respiration substrate during the later stages of the vase life when the carbohydrate content of the tissue is reduced. GREWAR (1973) and BLACKMAN (1953) came to a similar conclusion. Some of these amino acids, such as aspartic acid are common respiratory substrates (RAMAKRISHNA et aI., 1973). According to BRUSZEWSKI (1967) the carbohydrate concentration decreased noticeably during the later stages of the vase life of carnations. When the oxaloacetic acid concentration is lowered by catabolism during the Krebs cycle, and this cannot be fully compensated for by synthesis from carbohydrate reserves, this may result in reduced aspartic acid levels. Prote!n synthesis may, however, contribute to the reduction in concentration levels of certain amino acids. The synthesis of degrading enzymes can be expected during the later stages of ageing as found by ZAUBERMANN and SCHIFFMANN-NADEL (1972), HOBSON (1963) and HALL (1964) in ripening fruit. ATKIN and SRIVASTAVA (1970) reported a continuous protein synthesis despite a decreased protein content. Proteolysis and protein synthesis are likely concurrent processes.
Z. Pf1anzenphysiol. Bd. 86. S. 125-134. 1978.
Free amino acids in the petals of carnations
133
The observed increase in the ammonia concentration may lead to a decrease in the aspartic and glutamic acid concentration as found by BARASH et al. (1974) during the assimilation of ammonia by excised oat leaves. Wilting and the consequent decrease in oxygen availibility in tissues could stimulate synthesis of a-alanine and ),-aminobutyric acid, whilst inhibiting the synthesis of other amino acids such as aspartic acid (THOMPSON et al., 1966). Arginine may serve as an important nitrogen storage compound in carnation petals as found by HILL-COTTINGHAM and LLOYD-JONES (1973) in certain fruit trees. Certain amino acids, expecially those with common metabolic precursors, may be synthesized at the expense of each other. Feedback inhibition, as well as inhibition by other amino acids, may play an important role in the observed decrease in free amino acids. The loss of cell integrity during the later stages of vase life may drastically influence the relative concentration of protein and amino acids.
It can be concluded that certain amino acids accumulate during vase life mainly as result of nett synthesis and proteolysis. On the other hand a decrease in the concentration of other amino acids, especially near the end of vase life, is apparent. This is probably due to their utilization as respiratory substrates and their role in the synthesis of degrading enzymes.
References ATKIN, R. K. and B. I. S. SRIVASTAVA: Studies on protein synthesis by senescing and kinetin-treated barley leaves. Physiol. Plant. 23, 304-315 (1970). BARASH, I., T. SADON, and H. MOR: Relationship of glutamate dehydrogenase levels to free amino acids, amides and ammonia in excised oat leaves. Plant Cell Physiol. 15, 563-566 (1974). BARNETT, N. M. and A. W. NAYLOR: Amino acid and protein metabolism in Bermuda grass during water stress. Plant Physiol. 41, 1222-1230 (1966). BLACKMAN, F. F.: Respiratory drifts. In: James, W. O. (Ed.): Plant respiration, 40-81, Oxford at the Clarendon Press, London, 1953. BRUSZEWSKI, T. E.: The influence of chemical preservatives on postharvest respiration rates and biochemical changes in carnations (Dianthus caryophyllus L.). Ph. D.-thesis. The Pennsylvania State University, Biochemistry, 1967. CHEN, D., B. KESSLER, and S. P. MONSELISE: Studies on water regime and nitrogen metabolism of Citrus seedlings grown under water stress. Plant Physiol. 39, 379-386 (1964). GREWAR, L.: Biochemiese vcranderinge van sekere verbindings in kroonblare van verouderende angcliere. M. Sc.-thesis Rand Afrikaanse University, Johannesburg, 1973. HALL, C. B.: Cellulase activity in tomato fruits according to portion and maturity. Bot. Gaz. 125,156-157 (1964). HILL-COTTINGHAM, D. G. and C. P. LLOYD-JONES: Metabolism of carbon-14 labelled arginine, citrulline and ornithine in intact apple stems. Physiol. Plant. 29, 125-128 (1973). HOBSON, G. E.: Pectinesterase in normal and abnormal tomato fruit. Biochem. J. 86, 358-365 (1963). KEMBLE, A. R. and H. T. MACPHERSON: Liberation of amino acids in perennial rye grass during wilting. Biochem. J 58, 46-49 (1954). LIEIlERMAN, M., A. T. KUNISHI, and D. A. WARDALE: Ethylene production from methionine. Biochem. J. 97, 449-459 (1965). Z. Pjlanzenphysiol. Bd. 86. S. 125-134. 1978.
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MOTHES, K.: Der EinfluB des Wasserzustandes auf Fermentprozesse und Stoffumsatz. In: Ruhland, W. (Ed.), Encyclopedia of plant physiology 3, 656-664, Springer-Verlag, Berlin, 1956. - Ammoniakentgiftung und Aminogruppenvorrat. In: Ruhland, W. (Ed.), Encyclopedia of plant physiology 8,716-762, Springer-Verlag, Berlin, 1958. NGUYEN, S. T. and R. PAQUIN: Methodes d'extraction et de purification des acides amines libres et des proteines de tissus vegetaux. J. Chromatogr. 61, 349-351 (1971). NICHOLS, R.: Ethylene production during senescence of flowers. J. hortic. Sci. 41, 279-290 (1966). RAMAKRISHNA, M., D. B. WANKHEDE, and M. R. RAGHAVENDRA RAO: Incorporation of U- 14 C aspartate into lysine, threonine and other amino acids in bengal gram(Cicer arietinum). Indian J. biochem. biophys. 10, 285-286 (1973). SAKAI, S. and H. IMASEKI: Ethylene biosynthesis: Methionine as an in vivo precursor of ethylene in auxin-treated mungbean hypocotyl segments. Planta (Berlin) 105, 165-173 (1972). SCHOBERT, B.: The influence of water stress on the metabolism of diatoms.!' Osmotic resistance and proline accumulation in Cyclotella meneghiniana, Z. Pflanzenphysiol. 74, 106-120 (1974). SPENCER, P. W. and J. S. TITUS: Apple leaf senescence: Leaf disc compared to attached leaf. Plant Physiol. 51, 89-92 (1973). THOMPSON, J. F., C. R. STEWART, and C. J. MORRIS: Changes in amino acid content of excised leaves during incubation. 1. The effect of water content of leaves and atmospheric oxygen level. Plant Physiol. 41, 1578-1584 (1966). VAN DER WESTHUIZEN, A. J.: Veranderinge in vrye en gebonde aminosuurkonsentrasies in verouderende angelierkroonblare. M.sc.-thesis, Rand Afrikaans University, Johannesburg, 1976. WALDREN, R. P., 1. D. TEARE, and S. W. EHLER: Changes in free proline concentration in sorghum and soybean plants under field conditions. Crop. Sci. 14, 447-450 (1974). WEINSTEIN, L. H.: Senescence of roses.!. Chemical changes associated with senescence of cut «Better Times» roses. Contrib. Boyce Thompson Inst. Plant Res. 19, 33-48 (1957). ZUABERMAN, G. and M. SCHIFFMANN-NADEL: Pectin methylesterase and polygalacturonase in avocado fruit at various stages of development. Plant Physiol. 49, 864-865 (1972).
Mr. A. J. VAN DER WESTHUIZEN, Institute for Botanical Research, University of Pot chefstroom, Potchefstroom, Republic of South Africa.
Z. PJlanzenphysiol. Bd. 86. S. 125-134. 1978.