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Glycolate Content, Glycolate Oxidase and Catalase Activity in Intact Sunflower Plant During Ageing and Development
Biochem. Physiol. Pflanzen 175,23-28 (1980)
Glycolate Content, Glycolate Oxidase and Catalase Activity in Intact Sunflower Plant During Ageing and Development UDAYAN SARKAR and M. A. CHOUDHURI Department of Botany, Burdwan University, Burdwan , India Key Term Index: glycolate, glycolate oxidase, catalase, ageing, developmental; Helianthus anl1u1ts.
Summary Glycolate accumulation and activities of glycolate oxidase and catalase in the 1st, 7th, 15th and the last leaf of intact sunflower plant (H elianthus annuus L.) were studied in relation to its developmental stages. At the vegetative stage, glycolate accumulation gradually increased with the advance in leaf age. This trend was disturbed at the flowering stage when maximum accnmulation occurred in the 1st leaf and minimum in the 7th. At the fruiting stage, the same trend as shown in the vegetative stage was obtained, though the contents were much higher. The glycolate oxidase activity at the vegetative stage gradually increased with leaf age but declined to a minimal level in the last leaf. During flowering stage, this activity was maximum in the 1st leaf, then declined with age. The activity of this enzyme reduced to a lowest level in the 1st leaf at the fruiting stage and declined in older leaves. Catalase activity was lower in the 1st leaf alld higher in the 7th leaf at all the three stages of develop· ment. This activity decreased in the 15th leaf at both the vegetative and fruiting stages but increased at the flowering stage. The activity of this enzyme always remained lower in the last leaf.
Introduction
Photo respiration is characterized by oxidation of glycolate synthesized as an early product of photosynthesis and glycolate oxidase is known as the key enzyme for the initial oxidation of glycolate, the photorespirotory substrate (ZELITCH 1971). Catalase is another important enzyme of the glycolate pathway which catalyses the breakdown of H 20 2 produced during the oxidation of glycolate (TOLBERT 1971). There are several lines of evidence indicating the involvement of glycolate metabolism in photorespiration and photosynthesis. ZELITCH (1973) pointed out that the synthesis of glycolate and its oxidation to CO 2 diminish net CO 2 assimilation during photosynthesis. It has also been demonstrated that under conditions of reduced photorespiration less glycolate is synthesized in leaves of soybean and sunflower (SERVAITES and OGREN 1977; ZELITCH 1973). The decline in the activity of glycolate oxidase may lead to an accumulation of glycolic acid, provided the synthetic ability of the glycolate producing systems is not concomitantly reduced. Reports concerning the glycolate metabolism in sunflower plants are sporadic and fragmentary (SALIN and HOMANN 1973). No attempt has yet been made to study the glycolate metabolism in sunflower plant during ageing and development of the plant. Thus the pattern of glycolate metabolism during ageing of an intact C3 plant like sunflower will be an interesting study. The present study was,
24
U. SARKAR and M. A. OHOUDHURI
therefore, aimed to analyse the accumulation of glycolate as well as the activities of glycolate oxidase and catalase in the leaves of different ages of intact sunflower plant at three developmental stages, viz., vegetative, flowering and fruiting. Material and Methods Plant material Glycolate content and the activity of the two enzymes viz., glycolate oxidase and catalase were analysed in the 1st, 7th, 15th and the last (oldest) leaf (from the apex to base) of sunflower plant (Helianthus annuus L. Ov. EO 68414) during each of the three growth stages viz., vegetative (32-46 days), flowering (54-62 days) and fruiting (76-83 days). The plants were grown in 6 x 2 m piots with a spacing of 30 cm in between plants and 60 cm in between the rows. Randomized leaf samples from leaves of each category of plants mentioned above were separately collected from 10 plants and the following parameters were studied.
Determination of glycolate content Extraction, isolation and colorimetric determination of glycolate were done by the method of ZELITCH (1958). The glycolic acid was extracted by homogenizing 1 g of tissue with 5 ml of 0.01 M NaHS0 3 (previously heated at approx. 90 °0). The homogenate was centrifuged at 18,000· g for 20 min. The residue was stirred with about 10 ml of water and the centrifugation was repeated. The combined supernates (approx. 25 ml) were used for the separation of glycolic acid on a Dowex-1 x 10acetate anion exchange column; the estimation of glycolic acid content was done by developing pink colour with 0.01 % 2,7-dihydroxynapththalene in conc. H2 S0 4 (wJv) and measuring the intensity at 540 nm in a Spectrochem spectrophotometer. The glycolate content was calculated from a standard curve.
Determination of glycolate oxidase activity Glycolate oxidase was extracted from 0.5 g leaf tissue by homogenizing with 3 ml of 50 mM Tris-HOI buffer (pH 8) containing 5 mM MgOI 2 , 5 mM 2-mercaptoethanol and 1 mM EDTA at 0 °0, and centrifuged at 10,000· g for 20 min (KUNDU et al. 1976). The pigments were removed by adsorbing in activated charcoal for 10 min. The resultant volume was made up to 7 ml with the extraction buffer and was used as an enzyme source. The activity of glycolate oxidase was measured according to the method of OSMOND and HARRIS (1971) with slight modifications. The assay mixture contained Tris-HOI buffer (pH 7.8), 0.1 mM flavin mononucleotide (2 ml), 10 mM phenyl-hydrazine-hydrochloride (1 ml), 5 mM of glycolate (2 ml) and 2 ml of enzyme solution. The reaction mixture was incubated at 35 °0 for 10 min and the absorbance was measured at 340 nm in a Spectrochem spectrophotometer.
Determination of catalase activity The extraction of the enzyme catalase and the measurement of its activity were done according to the method described by BISWAS and OHOUDHURI (1978). The enzyme was extracted from 0.1 g leaf tissue with 3 ml of 0.1 M phosphate buffer (pH 6.5) at 0 °0. The homogenate was centrifuged at 10,000· g for 25 min, the supernate was made up to 7 ml with the buffer and used as enzyme source The reaction mixture for the assay of this enzyme contained 1 ml of enzyme solution and 1 ml 0.0025 M H 2 0 2 and was incubated for 10 min at 37 °0. The reaction was stopped by adding 3 ml 0.8 % titanyl sulphate in 25 % H 2 S04 (vJv), and the colour was read at 420 nm. In each case of enzyme assay, zero time control was taken as blank and the activity of each enzyme was expressed as [(LJ AxTv)JTxv], where A is the absorbance of the sample after incubation minusthe absorbance at zero time control, Tv is the total volume of the filtrate, t is the time (min) of incubation with the substrate and v is the total volume of the filtrate taken for incubation (FICK and QUALSET 1975).
25
Glycolate Metabolism in Ageing Sunflower
Results
Table 1 shows that glycolate content is minimum in the 1st feaf and maximum in the last leaf, while the 7th and the 15th leaf showed intermediate values at the vegetative stage. During flowering stage, this picture changed and maximum amount of glycolate was recorded by the 1st leaf subtending the flower. The amount of glycolate dropped to a minimum level in the 7th leaf and rose to about two-fold in the 15th and in the last leaf (partially senesced at this stage). At the fruiting stage, the glycolate content of Table 1. Glycolate content (nmole/g fresh weight) in different aged leaves during three stages of development. Glycolate content of the 1st, 7th, 15th and the last leaf of intact sunflower plant during vegetative, flowering and fruiting stages were measured Leaf position (from apex)
1st 7th 15th Last
Glycolate content of leaves at growth stages Vegetative (32-46 days)
Flowering (54-62 days)
Fruiting (76-83 days)
S.E.
S.E.
S.E.
0.330 1.061 1.061 1. 78
± 0.0054 ± 0.0090 ± 0.0098
± 0.0308
5.61 2.31 4.22 4.49
± 0.0853 ± 0.0594 ± 0.0670 ± 0.0460
3.63 3.76 4.30 5.50
± 0.0702 ± 0.0850 ± 0.0904 ± 0.0813
the 1st leaf dropped substantially compared to that at the flowering stage. At this stage of development maximum accumulation of glycolate was encountered in the last leaf. There was a gradual increase in the contents of glycolate in the leaves with increasing age (Table 1). The activities of glycolate oxidase in different aged leaves of sunflower at three growth stages have been shown in Table 2. Glycolate oxidase activity gradually increased from 1st leaf to 15th leaf and then declined to a minimum level in the last leaf during the vegetative stage. At the flowering stage, glycolate oxidase activity was maximum in the 1st leaf and then decreased with age of the leaf. During the fruiting stage, the activity of glycolate oxidase was reduced to a lowest level. The activity of this enzyme Table 2. Activity of glycolate oxidase in different aged leaves during three developmental stages. The enzyme activity was expressed as (,1 Ax Tv/txv) and measured in the 1st, 7th, 15th and the last leaf of intact sunflower plant during vegetative, flowering and fruiting stages Leaf position (from apex)
1st 7th 15th Last
Activity of enzyme Vegetative (32-46 days)
Flowering (54-62 days)
Fruiting (76-83 days)
S.E.
S.E.
S.E.
10.5 11.6 17.5 8.0
± 0.505
± 0.650
± 0.741 ± 0.360
17.5 ± 0.763 1.1850 10.5 10.0 ± 0.779 7.5 ± 0.300
±
0.7 ± 0.021 10.5 ± 0.779 6.8 ± 0.153 6.6 ± 0.234
26
U.
SARKAR
and :\1. A.
CHOUDHURI
increased considerably in the 7th leaf and then declined with further advance in age of the leaf. The catalyse activity at the vegetative stage was minimum in the 1st leaf, then increased in the 7th leaf but decreased with further increase in the age of the leaf (Table 3). The activity of this enzyme at the flowering stage gradually increased with feaf age. The catalase activity of the last leaf (partially senesced) was, however, negligibly small. The activity of this enzyme at the fruiting stage was lowest in the last leaf (partially senesced) and highest in the 7th leaf. Both the 1st and the 15th leaf showed an intermediate activity of this enzyme (Table 3). Table 3. Activity of catalase in different aged leaves during three developmental stages. The enzyme activity was expressed as (Ll AxTvftxv) and measured in the 1st, 7th, 15th and the last leaf of intact sunflower plant during vegetative, flowering and fruiting stages Leaf position (from apex)
1st 7th 15th Last
Activity of enzyme Vegetative (32 -46 days)
Flowering (54-62 days)
Fruiting (76-83 days)
S.E.
S.E.
S.E.
70 ± 1.0504 1043 ± 4.0415 1029 ± 0.2886 1018.5 ± 3.7288
133 ± 140 ± 154 ± 14 ±
1.893 1.343 0.500 0.929
56 ± 154 ± 42 ± 14 ±
0.862 1.100 0.723 0.736
Discussiou
Photorespiration has a close connection with synthesis and further oxidation of glycolic aeid (ZELITCH 1975). Thus the pattern of glycolate accumulation and the activities of glycolate oxidase and catalase in different aged leaves of sunflower will throw some light on the metabolic characteristics of glycolate in these leaves versus photorespiration. The present study shows that glycolate accumulation increased with leaf age at the vegetative stage. But this pattern of glycolate accumulation was disturbed at the flowering stage, when maximum accumulation of glycolate took place in the 1st leaf and minimum in the 7th leaf, though the older leaves showed higher levels of glycolic acid. The trend of increasing accumulation of glycolate in the leaves of increasing age was again achieved during the fruiting stage of the plant. The higher accumulation of glycolate in older leaves may be a reflection of its lower oxidation by glycolate oxidase. The highest accumulation of glycolate in the 1st leaf during flowering stage along with higher glycolate oxidase activity (discussed later) suggests an active metabolic status of this leaf at this stage. It should be noted that the 1st leaf subtends the flower and may be involved more intimately in the transport of materials to the reproductive parts. Lower accumulation of glycolate in the 1st leaf during fruiting stage may be explained on the basis of reduced synthetic activity and increased transport of metabolites to seeds (DROPTE and UPADRYAY 1975) resulting in partial senesceing condition of this
Glycolate Metabolism in Ageing Sunflower
27
leaf. Such a situation was also shown by BIswAs and CHOUDHURI (1978) in flag leaf of rice. The activity of glycolate oxidase further clarifies the different metabolic accumulation of glycolate in the leaves at three different growth stages. The activities of glycolate oxidase increased with leaf age. The oldest leaf showed a decline at the vegetative stage. This indicates that metabolism of glycolate is maximum in the older leaves than that in younger. But at the flowering stage maximum glycolate oxidase activity was recorded in the 1st leaf and then decreased with leaf age. Thus the picture was completely opposite to that shown at the vegetative stage. The glycolate oxidase activity of the 1st leaf at the fruiting stage was, however, minimum compared to that of older leaves. This may be ascribed to its partial senesceing condition at that stage of the plant. It has been shown by SALIN and HOMAKN (1978) that young tobacco leaves photorespire less than the older leaves and this difference is reflected by lower activities of photorespiratory enzymes in young leaves and an apparent inability to synthesize glycolate. KISAKI et al. (1973), on the other hand, showed that fastest photorespiration occurred in the younger leaves near the top of tobacco plant. The present study with different aged leaves of sunflower at the three developmental stages clearly indicates that the developmental status of the plant has a considerable influence on the metabolic pattern of the enzyme in leaves. It was demonstrated by KAR and MrSHRA (1976) that catalase activity decreases with leaf age. With the exception of the 1st leaf (which remains in an immature stage during vegetative stage), this trend was maintained by the leaves of sunflower plant only at the vegetative stage. During the flowering and fruiting stages the catalase activity was found to be lower in the 1st and the last leaf and this was presumably due to their senescing condition, while that of the 7th and the 15th leaf, which were perhaps metabolically more active during this period, remained at a higher level. Thus the present study provides evidence that glycolate metabolism with respect to glycolate accumulation and activities of glycolate oxidase and catalase considerably varies in leaves of different ages and the developmental status of the plant exerts modifying effects on such metabolic event of the plant. Acknowledgement One of the authors (U.S.) gratefully acknowledges the award of a Senior Research Fellowship by the Council of Scientific and Inuustrial Research, New Delhi, India.
References DrswAS, A. K., and CnOUDHURl, ~I. A.: Differential behaviour of the flag leaf of intact rice plant uuring ageing. Dioehem. Physiol. Pflanzen 173, 220 - 2:28 (1978). DUOPTE, A. M., and UP"\DHYAY, U. C.: Role of bracts and top six leaves in grain production in Sunflower. Ind. J. Plant Physiol. 18, 1-4 (1975). FrcK, N. G., and QUALSET, C. 0.: Genetic ('ontrol of endosperm amylase activity and gibberellin responses in standard-height and short-sattured wheat. Proc. Natl. Acad. Sci. USA 72, 892-895
(1975).
28
U. SARKAI{ and M. A. CHOUDHURI, Glycolate Metabolism in Ageing Sunflower
KAR, M., and MISHRA, D.: Catalase, peroxidase, and polyphenol oxidase activities during rice leaf senescencne. Plant Physiol. 57, 315-319 (1976). KIsAKI, T., HIRABAYASHI, S., and YANO, N.: Effect of the age of tobacco leaves on photosynthesis and photorespiration. Plant Cell Physiol. 14, 505-504 (1973). Kmmu, A., PALIT, P., MANDAL, R. K., and SIRCAR, S. :JiJ.: C3-Type photosynthetic carbon dioxide fixation in rice plant. Plant Biochem. J. 3, 111-118 (1976). OS:lWND, C. E., and HARRIS, B.: Photorespiration during photosynthesis. Biochem. Biophys. Acta. 234,270-282 (1971). SALIN, ~L 1., and HOMANN, P. H.: Glycolate metabolism in young and old tobacco leaves, and effects of o:-hydroxy-2-pyridinemethane sulfonic acid. Can. J. Bot. 51, 1857 -1856 (1973). SERVAITES, J. C., and OGREN, W. L.: Chemical inhibition of glycolate pathway in soybean leaf cells. Plant. Physiol. 60, 461- 466 (1977). TOLBERT, N. E.: Microbodies-peroxisomes and glyoxysomes. Annu. Rev. Plant. Physiol. 22, 45-74 (1971). ZELITCH, I.: The role of glycolic acid oxidase in the respiration of leaves. J. BioI. Chern. 233, 12991303 (1958). Photosynthesis, photorespiration, and Plant Productivity. Academic Press, New York 1971. Alternate pathways of glycolate synthesis in Tobacco and Maize leaves in relation to rates of photorespiration. Plant Physiol. ill, 299-305 (1973). Improving the efficiency of photosynthesis. Science 188, 626-633 (1975). Received July 16, 1979.
Authors' address: UDAYAN SARKAR and M. A. CHOUDIIL'RI, Plant Physiology and Biochemistry Laboratory, Botany Department, Burdwan University, Burdwan 713104, India.
Glycolate Content, Glycolate Oxidase and Catalase Activity in Intact Sunflower Plant During Ageing and Development UDAYAN SARKAR and M. A. CHOUDHURI Department of Botany, Burdwan University, Burdwan , India Key Term Index: glycolate, glycolate oxidase, catalase, ageing, developmental; Helianthus anl1u1ts.
Summary Glycolate accumulation and activities of glycolate oxidase and catalase in the 1st, 7th, 15th and the last leaf of intact sunflower plant (H elianthus annuus L.) were studied in relation to its developmental stages. At the vegetative stage, glycolate accumulation gradually increased with the advance in leaf age. This trend was disturbed at the flowering stage when maximum accnmulation occurred in the 1st leaf and minimum in the 7th. At the fruiting stage, the same trend as shown in the vegetative stage was obtained, though the contents were much higher. The glycolate oxidase activity at the vegetative stage gradually increased with leaf age but declined to a minimal level in the last leaf. During flowering stage, this activity was maximum in the 1st leaf, then declined with age. The activity of this enzyme reduced to a lowest level in the 1st leaf at the fruiting stage and declined in older leaves. Catalase activity was lower in the 1st leaf alld higher in the 7th leaf at all the three stages of develop· ment. This activity decreased in the 15th leaf at both the vegetative and fruiting stages but increased at the flowering stage. The activity of this enzyme always remained lower in the last leaf.
Introduction
Photo respiration is characterized by oxidation of glycolate synthesized as an early product of photosynthesis and glycolate oxidase is known as the key enzyme for the initial oxidation of glycolate, the photorespirotory substrate (ZELITCH 1971). Catalase is another important enzyme of the glycolate pathway which catalyses the breakdown of H 20 2 produced during the oxidation of glycolate (TOLBERT 1971). There are several lines of evidence indicating the involvement of glycolate metabolism in photorespiration and photosynthesis. ZELITCH (1973) pointed out that the synthesis of glycolate and its oxidation to CO 2 diminish net CO 2 assimilation during photosynthesis. It has also been demonstrated that under conditions of reduced photorespiration less glycolate is synthesized in leaves of soybean and sunflower (SERVAITES and OGREN 1977; ZELITCH 1973). The decline in the activity of glycolate oxidase may lead to an accumulation of glycolic acid, provided the synthetic ability of the glycolate producing systems is not concomitantly reduced. Reports concerning the glycolate metabolism in sunflower plants are sporadic and fragmentary (SALIN and HOMANN 1973). No attempt has yet been made to study the glycolate metabolism in sunflower plant during ageing and development of the plant. Thus the pattern of glycolate metabolism during ageing of an intact C3 plant like sunflower will be an interesting study. The present study was,
24
U. SARKAR and M. A. OHOUDHURI
therefore, aimed to analyse the accumulation of glycolate as well as the activities of glycolate oxidase and catalase in the leaves of different ages of intact sunflower plant at three developmental stages, viz., vegetative, flowering and fruiting. Material and Methods Plant material Glycolate content and the activity of the two enzymes viz., glycolate oxidase and catalase were analysed in the 1st, 7th, 15th and the last (oldest) leaf (from the apex to base) of sunflower plant (Helianthus annuus L. Ov. EO 68414) during each of the three growth stages viz., vegetative (32-46 days), flowering (54-62 days) and fruiting (76-83 days). The plants were grown in 6 x 2 m piots with a spacing of 30 cm in between plants and 60 cm in between the rows. Randomized leaf samples from leaves of each category of plants mentioned above were separately collected from 10 plants and the following parameters were studied.
Determination of glycolate content Extraction, isolation and colorimetric determination of glycolate were done by the method of ZELITCH (1958). The glycolic acid was extracted by homogenizing 1 g of tissue with 5 ml of 0.01 M NaHS0 3 (previously heated at approx. 90 °0). The homogenate was centrifuged at 18,000· g for 20 min. The residue was stirred with about 10 ml of water and the centrifugation was repeated. The combined supernates (approx. 25 ml) were used for the separation of glycolic acid on a Dowex-1 x 10acetate anion exchange column; the estimation of glycolic acid content was done by developing pink colour with 0.01 % 2,7-dihydroxynapththalene in conc. H2 S0 4 (wJv) and measuring the intensity at 540 nm in a Spectrochem spectrophotometer. The glycolate content was calculated from a standard curve.
Determination of glycolate oxidase activity Glycolate oxidase was extracted from 0.5 g leaf tissue by homogenizing with 3 ml of 50 mM Tris-HOI buffer (pH 8) containing 5 mM MgOI 2 , 5 mM 2-mercaptoethanol and 1 mM EDTA at 0 °0, and centrifuged at 10,000· g for 20 min (KUNDU et al. 1976). The pigments were removed by adsorbing in activated charcoal for 10 min. The resultant volume was made up to 7 ml with the extraction buffer and was used as an enzyme source. The activity of glycolate oxidase was measured according to the method of OSMOND and HARRIS (1971) with slight modifications. The assay mixture contained Tris-HOI buffer (pH 7.8), 0.1 mM flavin mononucleotide (2 ml), 10 mM phenyl-hydrazine-hydrochloride (1 ml), 5 mM of glycolate (2 ml) and 2 ml of enzyme solution. The reaction mixture was incubated at 35 °0 for 10 min and the absorbance was measured at 340 nm in a Spectrochem spectrophotometer.
Determination of catalase activity The extraction of the enzyme catalase and the measurement of its activity were done according to the method described by BISWAS and OHOUDHURI (1978). The enzyme was extracted from 0.1 g leaf tissue with 3 ml of 0.1 M phosphate buffer (pH 6.5) at 0 °0. The homogenate was centrifuged at 10,000· g for 25 min, the supernate was made up to 7 ml with the buffer and used as enzyme source The reaction mixture for the assay of this enzyme contained 1 ml of enzyme solution and 1 ml 0.0025 M H 2 0 2 and was incubated for 10 min at 37 °0. The reaction was stopped by adding 3 ml 0.8 % titanyl sulphate in 25 % H 2 S04 (vJv), and the colour was read at 420 nm. In each case of enzyme assay, zero time control was taken as blank and the activity of each enzyme was expressed as [(LJ AxTv)JTxv], where A is the absorbance of the sample after incubation minusthe absorbance at zero time control, Tv is the total volume of the filtrate, t is the time (min) of incubation with the substrate and v is the total volume of the filtrate taken for incubation (FICK and QUALSET 1975).
25
Glycolate Metabolism in Ageing Sunflower
Results
Table 1 shows that glycolate content is minimum in the 1st feaf and maximum in the last leaf, while the 7th and the 15th leaf showed intermediate values at the vegetative stage. During flowering stage, this picture changed and maximum amount of glycolate was recorded by the 1st leaf subtending the flower. The amount of glycolate dropped to a minimum level in the 7th leaf and rose to about two-fold in the 15th and in the last leaf (partially senesced at this stage). At the fruiting stage, the glycolate content of Table 1. Glycolate content (nmole/g fresh weight) in different aged leaves during three stages of development. Glycolate content of the 1st, 7th, 15th and the last leaf of intact sunflower plant during vegetative, flowering and fruiting stages were measured Leaf position (from apex)
1st 7th 15th Last
Glycolate content of leaves at growth stages Vegetative (32-46 days)
Flowering (54-62 days)
Fruiting (76-83 days)
S.E.
S.E.
S.E.
0.330 1.061 1.061 1. 78
± 0.0054 ± 0.0090 ± 0.0098
± 0.0308
5.61 2.31 4.22 4.49
± 0.0853 ± 0.0594 ± 0.0670 ± 0.0460
3.63 3.76 4.30 5.50
± 0.0702 ± 0.0850 ± 0.0904 ± 0.0813
the 1st leaf dropped substantially compared to that at the flowering stage. At this stage of development maximum accumulation of glycolate was encountered in the last leaf. There was a gradual increase in the contents of glycolate in the leaves with increasing age (Table 1). The activities of glycolate oxidase in different aged leaves of sunflower at three growth stages have been shown in Table 2. Glycolate oxidase activity gradually increased from 1st leaf to 15th leaf and then declined to a minimum level in the last leaf during the vegetative stage. At the flowering stage, glycolate oxidase activity was maximum in the 1st leaf and then decreased with age of the leaf. During the fruiting stage, the activity of glycolate oxidase was reduced to a lowest level. The activity of this enzyme Table 2. Activity of glycolate oxidase in different aged leaves during three developmental stages. The enzyme activity was expressed as (,1 Ax Tv/txv) and measured in the 1st, 7th, 15th and the last leaf of intact sunflower plant during vegetative, flowering and fruiting stages Leaf position (from apex)
1st 7th 15th Last
Activity of enzyme Vegetative (32-46 days)
Flowering (54-62 days)
Fruiting (76-83 days)
S.E.
S.E.
S.E.
10.5 11.6 17.5 8.0
± 0.505
± 0.650
± 0.741 ± 0.360
17.5 ± 0.763 1.1850 10.5 10.0 ± 0.779 7.5 ± 0.300
±
0.7 ± 0.021 10.5 ± 0.779 6.8 ± 0.153 6.6 ± 0.234
26
U.
SARKAR
and :\1. A.
CHOUDHURI
increased considerably in the 7th leaf and then declined with further advance in age of the leaf. The catalyse activity at the vegetative stage was minimum in the 1st leaf, then increased in the 7th leaf but decreased with further increase in the age of the leaf (Table 3). The activity of this enzyme at the flowering stage gradually increased with feaf age. The catalase activity of the last leaf (partially senesced) was, however, negligibly small. The activity of this enzyme at the fruiting stage was lowest in the last leaf (partially senesced) and highest in the 7th leaf. Both the 1st and the 15th leaf showed an intermediate activity of this enzyme (Table 3). Table 3. Activity of catalase in different aged leaves during three developmental stages. The enzyme activity was expressed as (Ll AxTvftxv) and measured in the 1st, 7th, 15th and the last leaf of intact sunflower plant during vegetative, flowering and fruiting stages Leaf position (from apex)
1st 7th 15th Last
Activity of enzyme Vegetative (32 -46 days)
Flowering (54-62 days)
Fruiting (76-83 days)
S.E.
S.E.
S.E.
70 ± 1.0504 1043 ± 4.0415 1029 ± 0.2886 1018.5 ± 3.7288
133 ± 140 ± 154 ± 14 ±
1.893 1.343 0.500 0.929
56 ± 154 ± 42 ± 14 ±
0.862 1.100 0.723 0.736
Discussiou
Photorespiration has a close connection with synthesis and further oxidation of glycolic aeid (ZELITCH 1975). Thus the pattern of glycolate accumulation and the activities of glycolate oxidase and catalase in different aged leaves of sunflower will throw some light on the metabolic characteristics of glycolate in these leaves versus photorespiration. The present study shows that glycolate accumulation increased with leaf age at the vegetative stage. But this pattern of glycolate accumulation was disturbed at the flowering stage, when maximum accumulation of glycolate took place in the 1st leaf and minimum in the 7th leaf, though the older leaves showed higher levels of glycolic acid. The trend of increasing accumulation of glycolate in the leaves of increasing age was again achieved during the fruiting stage of the plant. The higher accumulation of glycolate in older leaves may be a reflection of its lower oxidation by glycolate oxidase. The highest accumulation of glycolate in the 1st leaf during flowering stage along with higher glycolate oxidase activity (discussed later) suggests an active metabolic status of this leaf at this stage. It should be noted that the 1st leaf subtends the flower and may be involved more intimately in the transport of materials to the reproductive parts. Lower accumulation of glycolate in the 1st leaf during fruiting stage may be explained on the basis of reduced synthetic activity and increased transport of metabolites to seeds (DROPTE and UPADRYAY 1975) resulting in partial senesceing condition of this
Glycolate Metabolism in Ageing Sunflower
27
leaf. Such a situation was also shown by BIswAs and CHOUDHURI (1978) in flag leaf of rice. The activity of glycolate oxidase further clarifies the different metabolic accumulation of glycolate in the leaves at three different growth stages. The activities of glycolate oxidase increased with leaf age. The oldest leaf showed a decline at the vegetative stage. This indicates that metabolism of glycolate is maximum in the older leaves than that in younger. But at the flowering stage maximum glycolate oxidase activity was recorded in the 1st leaf and then decreased with leaf age. Thus the picture was completely opposite to that shown at the vegetative stage. The glycolate oxidase activity of the 1st leaf at the fruiting stage was, however, minimum compared to that of older leaves. This may be ascribed to its partial senesceing condition at that stage of the plant. It has been shown by SALIN and HOMAKN (1978) that young tobacco leaves photorespire less than the older leaves and this difference is reflected by lower activities of photorespiratory enzymes in young leaves and an apparent inability to synthesize glycolate. KISAKI et al. (1973), on the other hand, showed that fastest photorespiration occurred in the younger leaves near the top of tobacco plant. The present study with different aged leaves of sunflower at the three developmental stages clearly indicates that the developmental status of the plant has a considerable influence on the metabolic pattern of the enzyme in leaves. It was demonstrated by KAR and MrSHRA (1976) that catalase activity decreases with leaf age. With the exception of the 1st leaf (which remains in an immature stage during vegetative stage), this trend was maintained by the leaves of sunflower plant only at the vegetative stage. During the flowering and fruiting stages the catalase activity was found to be lower in the 1st and the last leaf and this was presumably due to their senescing condition, while that of the 7th and the 15th leaf, which were perhaps metabolically more active during this period, remained at a higher level. Thus the present study provides evidence that glycolate metabolism with respect to glycolate accumulation and activities of glycolate oxidase and catalase considerably varies in leaves of different ages and the developmental status of the plant exerts modifying effects on such metabolic event of the plant. Acknowledgement One of the authors (U.S.) gratefully acknowledges the award of a Senior Research Fellowship by the Council of Scientific and Inuustrial Research, New Delhi, India.
References DrswAS, A. K., and CnOUDHURl, ~I. A.: Differential behaviour of the flag leaf of intact rice plant uuring ageing. Dioehem. Physiol. Pflanzen 173, 220 - 2:28 (1978). DUOPTE, A. M., and UP"\DHYAY, U. C.: Role of bracts and top six leaves in grain production in Sunflower. Ind. J. Plant Physiol. 18, 1-4 (1975). FrcK, N. G., and QUALSET, C. 0.: Genetic ('ontrol of endosperm amylase activity and gibberellin responses in standard-height and short-sattured wheat. Proc. Natl. Acad. Sci. USA 72, 892-895
(1975).
28
U. SARKAI{ and M. A. CHOUDHURI, Glycolate Metabolism in Ageing Sunflower
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Authors' address: UDAYAN SARKAR and M. A. CHOUDIIL'RI, Plant Physiology and Biochemistry Laboratory, Botany Department, Burdwan University, Burdwan 713104, India.