Acta histochem. Bd. 56, S. 80-85 (1976)
Department of Botany, College of Basic Sciences, Punjab Agriculture University, Ludhiana, India
Studies on the physiology of pollen and pollen tube growth 1. Pinus roxburghii
By
MANJU MEHAN
and C. P.
MALIK
With 20 figures (Received July 15, 1975)
Summary Pollen grains and pollen tubes of Pinus roxburghii were subjected to histochemical technique with a view to determining the distributional pattern of reserve substances (ascorbic acid, proteins starch, pclysaccharides, nucleic acids) and some enzymes (acid phosphatase, glucose-d-phosphatase, peroxidase, esterase, cytochrome oxidase, succinate dehydrogenase). An attempt is made to correlate the activity of different enzymes in relation to pollen tube growth.
Recently, the study concerning localization of different enzymes and other substrates in pollen has received some attention (MALIK et aI., 1969; KNOX and HESLOPHARRISON, 1969; SOOD et aI., 1969; BREWBAKER, 1973; MALIK and MEHAN, 1974(1975). Such localization not only showed the region of pollen and pollen tube associated with particular enzymes but also helped in the understanding of the possible biochemical processes operative in that structure. The present study concerns with an investigation of differences in the activities of several enzymes, as revealed by histochemical procedures. These data are discussed in relation to morphogenesis of pollen tube.
Material and Methods The pollen grains were collected from Pinus roxburghii plants and germinated in liquid culture in non-corrosive cavity blocks. The culture medium consisted of 10 % sucrose, 100 mg!l boric acid 300 mg!l calcium chloride, 200 mg!l magnesium sulphate and 100 mg!l potassium nitrate. Cytochemical tests for general substances and enzymes were carried out according to the procedures of JENSEN (1962); CHAYEN et al. (1973); SHAYKH and ROBERTS (1974). The pollen germination occur red after 48 h of incubation.
Results and Discussion The localization pattern of general substances and enzymes in the pollen and pollen tubes is shown in Figures 1-20. Ungerminated pollen grains contained lipids, nucleic acid, abundant ascorbic acid, faint cytoplasmic polysaccharides, but lacked starch. There were several enzymes localized in the cytoplasm of the pollen; these included peroxidase, succinate dehydrogenase, cytochrome oxidase, glucose-6-phos-
81
Studies on the physiology, 1.
4
3
2
1
5
Figs. 1- 9. Distribution of general substances. Fig. 1. Polysaccharide distribution in the pollen and pollen tubes. N otice PAS cytoplasm.
+ reaction in
Figs. 2-3. Notice pectic substances on the wall of pollen tubes. Figs. 4 - 5. Observe intense deposition of ascorbic acid. Figs. 6
(,j-
9. Notice abundant starch grains in the pollen and pollen tubes cytoplasm.
Acta histochem. Ed. 56
the
82
1\1.
)IE H AN
and C. P .
MAI,IK
10
11
12
3
15 Figs. 10 - 20. L ocalization of en zymes . Figs. 10 -14. Observe loca liza t ion of ph osphoryl ase strong reaction in t he p ollen t u be t ips.
III
t he po llen grain and po llen t ub es . Mark
F ig. 15. Strong reaction fo r pe roxidase in t he po llen and pollen t ubes .
phatase, acid phosphat ase and esterase. Some of t hese (acid ph osphatase, esterase ) wer e localized in t he int ine of p ollen . The rea ction for most of t he enzy mes was st rong near the germinal slit regi on. Wh en incubated in basal medium, germinat ion of pollen began after 48 h. With the emergence of pollen tubes, starch gra ins appeared in the pollen and pollen tubes (Figs. 6-9) . The staining for polysaccharides, with PAS re-
Studies on the physiology, 1.
83
17
19 Fig. 16. Pollen cytoplasm showing RNA with Azure-B staining. Fig. 17-19. Succinate dehydrogenase activity in the pollen and pollen tubes. Observe strong reaction in the pollen tube tip.
action, also intensified in the cytoplasm and wall. Similarly, ascorbic acid also increased. At this stage, reaction for phosphorylase also appeared and gradually intensified (Figs. 10-14). The pollen tube cytoplasm had strong reaction for acid phosphatase, glucose-6- phosphatase, peroxidase, cytochrome oxidase and succinate dehydrogenase. The staining was intense in the extreme tip of the pollen tubes. In case of phosphorylase, the reaction granules were deep blue in colour. In general, the reaction for some enzymes was granular (acid phosphatase, succinate dehydrogenase) or diffused (peroxidase). 6*
84
M. MEHA~
and C. P.
MALIK
Fig. 20. Reaction for cytochrome oxidase expacially in the pollen tube tip.
Ripe and mature pollen grains were rich in ascorbic acid and same was true of pollen tubes. Pinus roxburghii is an anemophilaus plant and therefore, the viability of pollen spread over long period, was essential. Incidentally, pollination, fertilisation and maturity of female cones required a period of 3 years. The presence of ascorbic acid in all likelihood was concerned with the viability of pollen. Pinus pollen had no starch in its cytoplasm. When germinated in the sucrose medium, however, abundant starch was accumulated in the cytoplasm of pollen tubes. This suggested that in Pinus, pollen tube could undertake utility of exogenous sucrose and excess level of sugars could be accumulated as storage starch. This indicated the presence of enzymes which could metabolise sucrose into starch. The situation is comparable to Lilium but contrasted with Calotropis (MALIK and MEHAN, 1975). This was corroborated by high activity of phosphorylase in the pollen tubes. When incubated in water, the pollen failed to germinate and the activities of succinate dehydrogenase as well as cytochrome-oxidase were very low. The present studies have demonstrated that acid phosphatase and esterase were associated with the intine. These observations are in agreement with the observations of KNOX and HESLOP-HARRISON (1969). The reaction for acid phosphatase was predominantly localised in the cytoplasm of the pollen tube, especially in the extreme pollen tube tip. Presumably, this system was involved in either secretion processes or uptake. ASHFORD and MCCULLY (1970) demonstrated that acid phosphatase was involved in the lateral root differentation of plants. In the present study strong acid phosphatase activity was observed in the pollen tubes and this may suggest the involvement of this enzyme in the growth of pollen tubes. Glucose-6-phosphatase was localised intensely in the pollen tubes especially in the tip region. In fact, the staining pattern or-thiS" enzyme simulated that acid phosphatase. It is assumed that these enzymes function in active ion transport and possibly involved in polysaccharide bio-
of
Studies on the physiology, 1.
85
synthesis as well. TALJEDAHL (1969) has reported that glueose-6-phosphatase was a part of a glucoreception mechanism. Increased enzymatic activities during elongation of pollen tubes were observed for many enzymes e. g. cytochrome oxidase, succinate dehydrogenase and peroxidase. Succinate dehydrogenase and cytochrome oxidase were integrated into the processes of cellular respiration. The energy, thus, obtained was utilized in the elongation of the pollen tube. In the present study, it was found that peroxidase activity increased with the elongation of pollen tubes. This high enzyme activity decreased as the pollen tubes attained maturity. This enzyme may be involved in the pollen tube elongation by the destruction of some of the inhibitors which blocked metabolic processes conducive to the elongation of pollen tube.
Literature ASHFORD, A. E., and :VIcKuLLY, M. E., Local isation of naphthol AS-BI phosphatase activity in lateral and main root meristems of pea and corn. Protoplasma 70, 441- 456 (1970). BREWBAKER, J. L., Pollen enzymes and Isoenzymes in "Pollen: development and physiology" (ed.) by J. Heslop-Harrison. Butterworth, London 1973. CHAYEN, J., and BITENSKY, R. G., Practical Histochemistry. Willy & Co., London 1973. JENSEN, "V. A., Botanical Histochemistry, W. H. Freeman & Co., San Francisco 1962. KNOX, R. B., and HESLOP-HARRISON, J., Pollen wall enzymes. Taxonomic distribution and physical localisation in "Pollen: development and physiology", (ed.) by J. Heslop-Harrison, Butterworth, London 1973. MALIK, C. P., and MEHAN, MANJlY, Pollen Chemistry, enzymes and tube growth in Calotropis procera, 43rd Indian Society of Biological chemists. Abst ,; (1974). MALIK, C. P., and MEHAN, MANJU, Studies on the physiology of pollen and pollen tubes 2. Colotropis procera, Jour. Palyrology, 10,7-28. MALIK, C. P., TEWARI, H. B., and SOOD, P. P., On the functional significance of certain phosphatases in the germinating pollen grains of Portulaca grandiflora. Portug. Acta BioI. ll, 245 - 252 (1969). SHYKH, M. lYI., and ROBERTS, L. W., A histochemical study of phosphatases in root opical meristems. Ann. Bot. 38,165-174 (1974). SOOD, P. P., MALIK, C. P., and TEWARI, H. B., A histochemical study of localization of suceinate dehydrogenase in the germinating pollen grains of Eschscholtzia Californica. Biologia Plant. 14,260-272 (1969). TALJEDAHL, 1. B., Kinetics of glucose-6-phosphatase in pancreatic islets as revealed by staining histoehemistry- Histochemie 19, 335-362 (1969). Author's address: Prof. Dr. C. P. MALIK, Department of Botany, College of Basic Sciences, Punjab Agriculture University, Ludhiana, India.
Department of Botany, College of Basic Sciences, Punjab Agriculture University, Ludhiana, India
Studies on the physiology of pollen and pollen tube growth 1. Pinus roxburghii
By
MANJU MEHAN
and C. P.
MALIK
With 20 figures (Received July 15, 1975)
Summary Pollen grains and pollen tubes of Pinus roxburghii were subjected to histochemical technique with a view to determining the distributional pattern of reserve substances (ascorbic acid, proteins starch, pclysaccharides, nucleic acids) and some enzymes (acid phosphatase, glucose-d-phosphatase, peroxidase, esterase, cytochrome oxidase, succinate dehydrogenase). An attempt is made to correlate the activity of different enzymes in relation to pollen tube growth.
Recently, the study concerning localization of different enzymes and other substrates in pollen has received some attention (MALIK et aI., 1969; KNOX and HESLOPHARRISON, 1969; SOOD et aI., 1969; BREWBAKER, 1973; MALIK and MEHAN, 1974(1975). Such localization not only showed the region of pollen and pollen tube associated with particular enzymes but also helped in the understanding of the possible biochemical processes operative in that structure. The present study concerns with an investigation of differences in the activities of several enzymes, as revealed by histochemical procedures. These data are discussed in relation to morphogenesis of pollen tube.
Material and Methods The pollen grains were collected from Pinus roxburghii plants and germinated in liquid culture in non-corrosive cavity blocks. The culture medium consisted of 10 % sucrose, 100 mg!l boric acid 300 mg!l calcium chloride, 200 mg!l magnesium sulphate and 100 mg!l potassium nitrate. Cytochemical tests for general substances and enzymes were carried out according to the procedures of JENSEN (1962); CHAYEN et al. (1973); SHAYKH and ROBERTS (1974). The pollen germination occur red after 48 h of incubation.
Results and Discussion The localization pattern of general substances and enzymes in the pollen and pollen tubes is shown in Figures 1-20. Ungerminated pollen grains contained lipids, nucleic acid, abundant ascorbic acid, faint cytoplasmic polysaccharides, but lacked starch. There were several enzymes localized in the cytoplasm of the pollen; these included peroxidase, succinate dehydrogenase, cytochrome oxidase, glucose-6-phos-
81
Studies on the physiology, 1.
4
3
2
1
5
Figs. 1- 9. Distribution of general substances. Fig. 1. Polysaccharide distribution in the pollen and pollen tubes. N otice PAS cytoplasm.
+ reaction in
Figs. 2-3. Notice pectic substances on the wall of pollen tubes. Figs. 4 - 5. Observe intense deposition of ascorbic acid. Figs. 6
(,j-
9. Notice abundant starch grains in the pollen and pollen tubes cytoplasm.
Acta histochem. Ed. 56
the
82
1\1.
)IE H AN
and C. P .
MAI,IK
10
11
12
3
15 Figs. 10 - 20. L ocalization of en zymes . Figs. 10 -14. Observe loca liza t ion of ph osphoryl ase strong reaction in t he p ollen t u be t ips.
III
t he po llen grain and po llen t ub es . Mark
F ig. 15. Strong reaction fo r pe roxidase in t he po llen and pollen t ubes .
phatase, acid phosphat ase and esterase. Some of t hese (acid ph osphatase, esterase ) wer e localized in t he int ine of p ollen . The rea ction for most of t he enzy mes was st rong near the germinal slit regi on. Wh en incubated in basal medium, germinat ion of pollen began after 48 h. With the emergence of pollen tubes, starch gra ins appeared in the pollen and pollen tubes (Figs. 6-9) . The staining for polysaccharides, with PAS re-
Studies on the physiology, 1.
83
17
19 Fig. 16. Pollen cytoplasm showing RNA with Azure-B staining. Fig. 17-19. Succinate dehydrogenase activity in the pollen and pollen tubes. Observe strong reaction in the pollen tube tip.
action, also intensified in the cytoplasm and wall. Similarly, ascorbic acid also increased. At this stage, reaction for phosphorylase also appeared and gradually intensified (Figs. 10-14). The pollen tube cytoplasm had strong reaction for acid phosphatase, glucose-6- phosphatase, peroxidase, cytochrome oxidase and succinate dehydrogenase. The staining was intense in the extreme tip of the pollen tubes. In case of phosphorylase, the reaction granules were deep blue in colour. In general, the reaction for some enzymes was granular (acid phosphatase, succinate dehydrogenase) or diffused (peroxidase). 6*
84
M. MEHA~
and C. P.
MALIK
Fig. 20. Reaction for cytochrome oxidase expacially in the pollen tube tip.
Ripe and mature pollen grains were rich in ascorbic acid and same was true of pollen tubes. Pinus roxburghii is an anemophilaus plant and therefore, the viability of pollen spread over long period, was essential. Incidentally, pollination, fertilisation and maturity of female cones required a period of 3 years. The presence of ascorbic acid in all likelihood was concerned with the viability of pollen. Pinus pollen had no starch in its cytoplasm. When germinated in the sucrose medium, however, abundant starch was accumulated in the cytoplasm of pollen tubes. This suggested that in Pinus, pollen tube could undertake utility of exogenous sucrose and excess level of sugars could be accumulated as storage starch. This indicated the presence of enzymes which could metabolise sucrose into starch. The situation is comparable to Lilium but contrasted with Calotropis (MALIK and MEHAN, 1975). This was corroborated by high activity of phosphorylase in the pollen tubes. When incubated in water, the pollen failed to germinate and the activities of succinate dehydrogenase as well as cytochrome-oxidase were very low. The present studies have demonstrated that acid phosphatase and esterase were associated with the intine. These observations are in agreement with the observations of KNOX and HESLOP-HARRISON (1969). The reaction for acid phosphatase was predominantly localised in the cytoplasm of the pollen tube, especially in the extreme pollen tube tip. Presumably, this system was involved in either secretion processes or uptake. ASHFORD and MCCULLY (1970) demonstrated that acid phosphatase was involved in the lateral root differentation of plants. In the present study strong acid phosphatase activity was observed in the pollen tubes and this may suggest the involvement of this enzyme in the growth of pollen tubes. Glucose-6-phosphatase was localised intensely in the pollen tubes especially in the tip region. In fact, the staining pattern or-thiS" enzyme simulated that acid phosphatase. It is assumed that these enzymes function in active ion transport and possibly involved in polysaccharide bio-
of
Studies on the physiology, 1.
85
synthesis as well. TALJEDAHL (1969) has reported that glueose-6-phosphatase was a part of a glucoreception mechanism. Increased enzymatic activities during elongation of pollen tubes were observed for many enzymes e. g. cytochrome oxidase, succinate dehydrogenase and peroxidase. Succinate dehydrogenase and cytochrome oxidase were integrated into the processes of cellular respiration. The energy, thus, obtained was utilized in the elongation of the pollen tube. In the present study, it was found that peroxidase activity increased with the elongation of pollen tubes. This high enzyme activity decreased as the pollen tubes attained maturity. This enzyme may be involved in the pollen tube elongation by the destruction of some of the inhibitors which blocked metabolic processes conducive to the elongation of pollen tube.
Literature ASHFORD, A. E., and :VIcKuLLY, M. E., Local isation of naphthol AS-BI phosphatase activity in lateral and main root meristems of pea and corn. Protoplasma 70, 441- 456 (1970). BREWBAKER, J. L., Pollen enzymes and Isoenzymes in "Pollen: development and physiology" (ed.) by J. Heslop-Harrison. Butterworth, London 1973. CHAYEN, J., and BITENSKY, R. G., Practical Histochemistry. Willy & Co., London 1973. JENSEN, "V. A., Botanical Histochemistry, W. H. Freeman & Co., San Francisco 1962. KNOX, R. B., and HESLOP-HARRISON, J., Pollen wall enzymes. Taxonomic distribution and physical localisation in "Pollen: development and physiology", (ed.) by J. Heslop-Harrison, Butterworth, London 1973. MALIK, C. P., and MEHAN, MANJlY, Pollen Chemistry, enzymes and tube growth in Calotropis procera, 43rd Indian Society of Biological chemists. Abst ,; (1974). MALIK, C. P., and MEHAN, MANJU, Studies on the physiology of pollen and pollen tubes 2. Colotropis procera, Jour. Palyrology, 10,7-28. MALIK, C. P., TEWARI, H. B., and SOOD, P. P., On the functional significance of certain phosphatases in the germinating pollen grains of Portulaca grandiflora. Portug. Acta BioI. ll, 245 - 252 (1969). SHYKH, M. lYI., and ROBERTS, L. W., A histochemical study of phosphatases in root opical meristems. Ann. Bot. 38,165-174 (1974). SOOD, P. P., MALIK, C. P., and TEWARI, H. B., A histochemical study of localization of suceinate dehydrogenase in the germinating pollen grains of Eschscholtzia Californica. Biologia Plant. 14,260-272 (1969). TALJEDAHL, 1. B., Kinetics of glucose-6-phosphatase in pancreatic islets as revealed by staining histoehemistry- Histochemie 19, 335-362 (1969). Author's address: Prof. Dr. C. P. MALIK, Department of Botany, College of Basic Sciences, Punjab Agriculture University, Ludhiana, India.
