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Compounds Released from Incompatible Apple Pollen during in vitro Germination
Originalarbeiten . Original Papers
Institute of Botany, University of Bologna, Bologna, Italy
Compounds Released from Incompatible Apple Pollen during in vitro Germination A.
SPERANZA
and G. L.
CALZONI
With 5 figures Received July 16, 1979 . Accepted August 20,1979
Summary Compounds released from pollen tubes of gametophytically controlled apple cultivar were investigated. Pollen was able to regulate the pH of the medium by means of surfacelinked compounds, whose buffering power was evaluated. Proteins, carbohydrates, RNA and polyamines were released in the culture. A possible role of polyamines in fertilization processes is indicated, in support of the hypothesis of extracellular enzyme synthesis controlling pollen tube growth. Key words: pollen germination, pH changes, polyamines, fertilization.
Introduction Pollen of Rosaceae displays a gametophytic monofactorial control of self-incompatibility. A stylar inhibition being involved, it is possible that the pollen incompatibility factors are perhaps synthesized mainly during tube growth rather than pre-stored in the pollen cell-wall (HESLOP-HARRISON, 1977). With this in mind, we searched for some compounds, released during pollen germination of incompatible Malus domestica cultivar, which might be related to the growth regulation of the pollen tube.
Materials and Methods Pollen of Malus domestica (BORKH), cv. Starkrimson, was collected, managed and stored as previously described (CALZONI et al., 1979). Germination tests were performed in 250 ml flasks, constantly shaken (80 cycles/min) for 2 h at 30°C. Liquid medium contained 0.2 M sucrose, 20 ,ug/ml HsB03 and 300 ,ug/ml Ca(NO a)2' 4H20. Different pH values were obtained with 0.01 N HCl or NaOH; in some specified cases citric acid-NaHP0 4 (Mc Ilvai-
Z. Pflanzenphysiol. Bd. 97. S. 95-102. 1980.
96
A. SPERANZA and G. L. CALZONI
ne) buffer was used at 0.01 and 0.01 M respectively. Germination was recorded by photomicrographs after 120 min of incubation; microscopical control in order to check for burst pollen tubes in the culture was negative. Liquid medium was separated from pollen by filtration on Millipore filters (SSWP 04700 SS 3 p,). Filtered medium was stored at -20 DC, in some cases liophylized using an Edwards vacuum pump. Titration of medium was carried out with 0.01 N HCI or NaOH (Normex, Carlo Erba). Proteins released in germination medium were assayed according to LOWRY et ai. (1951). Kirby's method (KIRBY et aI., 1964) modified for plants by LI and ANDERSEN (1967), to extract and determine total RNA was used. Polyamines were detected by direct dansylation according to SMITH and BEST (1977), then dansyl-amines were separated by one-dimensional TLC on Kieselgel 60 plates with concentration zone, 0.25 mm layer thickness (Merck). Plates were run in cyclohexane/ethylacetate (3 : 2), followed immediately by spraying with triethanolamine/isopropanol (1 : 4) to enhance and stabilize the fluorescence. Quantitative analysis of fluorescent compounds was performed in a Jasco spectrofluorometer (excitation and emission wavelengths 365 and 505 nm respectively), and referred to fluorescence of standards run on the same plate. Carbohydrates were assayed in pollen eluate obtained with Coca's solution (COCA, 1922), by a-naphtol test modified according to KIRBY and SMITH (1974).
Results We thorougly re-examined our earlier observations on the ability of pollen to influence the external medium, in particular the pH (CALZONI et aI., 1979). 100 ;;!
z
Q ao
~ ~
0::
~
IGO
•
Buffered medium
.&
Unbuffered mpdium
z
Q40
~ 0:: ~
J:
1"20 I I
0
4
5
6 7 8 INITIAL pH Fig. 1: Per cent pH variations of culture medium and germination of Starkrimson pollen (1 mg/ml), after 120 min incubation at different initial pH, with and without buffer.
z. PJlanzenphysiol. Bd. 97. S. 95-102. 1980.
Compounds released from apple pollen
97
Fig. 1 shows the per cent of pH variation in buffered and unbuffered media; in both cases minimal variations and optimum pH for germination are strictly related; the highest pH changes are associated with the extreme pH values. Therefore, the pollen seems to exert a remarkable control over pH, because all the initial pH values of media become close and similar each other at pH range of 6.0-6.5. It is important to remark that the buffer could not keep the initial pH quite constant, but a low molarity was needed to avoid inhibitory ion effects. Fig. 2 shows that pH changes actually depend on pollen concentration. We may suppose that this pH control is probably due to an elution of substances from pollen cell-walls, rather than to a real activity of living pollen. This elution may be more or less high owing to the pH of medium . 00 f-
8OfI. B uttf'r~
60
mt"cilum
•
ot v.obl," pollf'n
4C f-
2Cf-
0
01
0.5
~
1 2 1" 1" POLLEN mg/ml
Fig. 2 : Per cent pH variations and germination related min of incubation at initial pH 4.
to
pollen concentration, after 120
Really, these pH variations recovered after 120 min of germination are almost established when pollen is suspended in the culture medium, with a gentle shaking of a few seconds. Namely, the highest pH changes occur at this time we have called «zero», since foregoing the real time of incubation at 30 Table 1 shows an attempt to evaluate this buffering power of filtered medium, referred to pure medium at three different pH. The buffering power is most probably due to various ionizable groups of protein-like compounds, easily elutable from pollen cell-walls.
ac.
Z. P/lanzenphysiol. Bd. 97. S. 95-102.1980.
98
A. SPERANZA and G. L. CALZON!
Table 1: Buffer power of germination medium (filtrate) of Starkrimson pollen. A change of one pH unit was considered. Pure medium, taken as reference, was titrated in the same pH interval as filtrate. (For «0» time of germination, see text.) Initial pH
Sample Filtrate Filtrate Pure medium Filtrate Filtrate Pure medium Filtrate Filtrate Pure medium
4.0 4.0 4.0 6.0 6.0 6.0 8.0 8.0 8.0
Germination (min)
Final pH
0 120
6.0 6.1
0 120
6.2 6.3
0 120
6.5 6.5
HCI
NaOH
(.uUml)
(.uUml)
4.5 11.2 3.2 7.5 6.2 5.7 13.7 12.5 8.5
4.5 16.8 3.5 7.0 17.8 4.4 9.4 16.5 6.5
00 0.7
Z40~~"-----:::!350 Z'~:W~2:10'5~--350=' zid Z75
31ia zao
275
~nm
00
b' :--:Z;!;:75~--'3:::!1ia Z~~0:-:;Z'=75"---""3"::!50 Zior.........Z;!;:75,.------=!J50 nm ~""zj;;75"-----:::!350 Z40 00
o
30
60
120 Minutes
Fig. 3: Levels of UV-absorbing materials released by Starkrimson pollen (1 mg/ml) germinating at different initial pH.
Z. Pjlanzenphysiol. Bd. 97. S. 95-102. 1980.
Compounds released from apple pollen
99
.. pH 4
200
• pH 6 • pH 8
:€150 C'I
;z,
Z W I-
0
!fl00
120
Fig. 4: Proteins released in culture medium at different initial pH by germinating pollen (1 mg/ml) of Starkrimson. Levels of UV -absorbing materials released during germination appear to gradually increase (Fig. 3), showing a more and more clear-cut maximum in the general region of proteins (270-280 nm). Nevertheless, a more appropriate assay (LOWRY et al., 1951) was carried out to determine the amounts of released proteins during germination (Fig. 4). Coca's solution (COCA, 1922) was employed to make evident carbohydrates in pollen eluate of our cultivar, so we have a very strong evidence for their presence with a-naphtol test (KIRBY et al., 1974). Moreover, on the basis of Tupf's studies on Nicotiana (Tupf et al., 1974), we tried to identify macromolecules as ribonucleic acids in filtered medium after pollen germination. Small amounts of RNA are present in the liquid medium: 1.4 ,ug/ml in media at both initial pH 4 and 6, and 3.4 ,ug/ml at pH 8. So far these data are indicative of total RNA, without any fractionation. Fig. 5 shows the recovery in pollen filtrate (initial pH 6) of other molecules of great biological importance, like polyamines. They appear to be present at the first stages of germination, then gradually increase, except spermine that shows a very large increment between 60 and 120 min. Moreover, a further interesting issue is observed in TLC plates, where strongly fluorescent compounds with a very low Ram are present. According to DION and HERBST (1970) they are interpretable as
z. PJlanzenphysiol. Bd. 97. S. 95-102. 1980.
100
A. SPERANZA and G. L. CALZONI 4r---------------~-.
.. Sm
:€(5
•
Pu
•
Sd
E3 C
if)
W Z
~
~2
0
CL
o
30
60
MINUTES
120
Fig. 5: Polyamines released by germinating pollen (2 mg/ ml) of Starkrimson, at initial pH 6.
acetyl-polyamines, although quantitative analysis was not made for unavailability of corresponding standards. Acetyl-polyamines are believed to playa role in the control of RNA synthesis (BACHRACH, 1973). Discussion Our data on Malus domestica pollen point to a strong buffering power of its surface-linked compounds. Such control of external pH may also be important in vivo, because it should support pollen germination even when not neutral treatments are carried out in blossoming. Opinions conflict as to whether pH influences pollen germination (VASIL and BOSE, 1959); our data may indicate (Fig. 1) a clear relationship between pH and pollen germination if only a buffer system is present. On the other hand, without any buffer system, germination actually seems to be almost independent of initial pH in the culture medium. An other interesting point is the carbohydrate recovery in pollen eluate of Starkrimson; in this respect, we hypothesize their possible linkage with proteins. In fact, the association of proteins with carbohydrates in pollen cell-wall would
Z. P/lanzenphysiol. Ed. 97. S. 95-102. 1980.
Compounds released from apple pollen
101
facilitate their extraction as glycoproteins (STANLEY and LINSKENS, 1974). According to LEWIS model of gametophytic self-incompatibility (1960), the inhibition factors arise from complexed glycoproteins on the surface of pollen tubes. Concerning the released ribonucleic acid, it is important to remark that RNA could be necessary for an extracellular protein synthesis as supposed by Tupf et al. (1974). In Nicotiana pollen culture (Tupf et al., 1974), fractionation on MAK column indicates rRNA and tRNA in the medium; in addition, the rapid movement of amino acids from pollen tubes (LINSKENS and SCHRAUWEN, 1969) and the occurrence of a new protein in the medium (Tupf, 1963) suggest this possibility. Molar ratio spermidine/spermine is very low if rapported to the inside content of ungerminated pollen of Starkrimson (BAGNI et al., 1978) (SD/SM = 35.5). This fact probably reflects a large polyamine synthesis in growing pollen tubes; this problem is still under ir_vestigation. Polyamines are known to promote cell division in plant and animal tissues. They playa role in almost every step of RNA and protein synthesis; owing to their basic groups they are able to bind with acidic groups, expecially of nucleic acids (BAGNI and SERAFINI-FRACASSINI, 1974). Polyamine recovery in pollen culture medium may suggest a possible role in the pro gamic phase of fertilization processes. Namely, pollen growth in incompatible and compatible systems may depend on released compounds interacting with the stylar tissues. To this purpose, the hypothesis of extracellular protein synthesis may be of great importance, so giving a real significance to polyamines released from pollen tubes. Acknowledgements This work was supported by the Consiglio Nazionale delle Ricerche, «Progetto Finalizzato Biologia della Riproduzione». The authors wish to thank Prof. N. BAGNI for critical reading of the manuscript; they are also indebted with the Defendi Farm for supplying tree branches, and with Dr. G. CIAFARDINI for lyophilizing some materials.
References BACHRACH, U.: In: Function of Naturally Occurring Polyamines, p. 106. Acad. Press, New York and London, 1973. BAGNI, N. and D. SERAFINI FRACASSINI: In: Plant Growth Substances, p. 1205. Hirokawa Pub!. Co. Inc., Tokyo, 1974. BAGNI, N., D. SERAFINI FRACASSINI, V. R. VILLANUEVA, and R. C. ADLAKA: Riv. Ortoflorofrutt. Ita!. 62, 470 (1978). CALZONI, G. L., A. SPERANZA, and N. BAGNI: Scientia Hort. 10,49 (1979). COCA, A. F.: J. Immuno!. 7, 163 (1922). DION, A. S. and E. J. HERBST: Ann. New York Acad. Sci. 171, 723 (1970). HESLOP-HARRISON, J.: In: Incompatibility in Angiosperms, p. 82. Springer-Verlag, Berlin, Heidelberg, New York, 1977.
Z. Pjlanzenphysiol. Bd. 97. S. 95-102. 1980.
102
A. SPERANZA and G. L. CALZONI
KIRBY, E. G. and J. E. SMITH: In: Fertilization in Higher Plants, p. 127. North-Holland Pub!. Co., Amsterdam, 1974. KIRBY, K. S.: In: Progress in Nucleic Acid Research, p. 2. Acad. Press, New York and London, 1964. LEWIS, D.: Proc. Roy. Soc. London, Ser. B, 151, 468 (1960). LI, P. H. and W. R. ANDERSEN: Nature 214,86 (1967). LINSKENS, H. F. and J. SCHRAUWEN: Acta Bot. Neerl. 18, 605 (1969). LOWRY, O. H., N. J. ROSEBROUGH, A. LEWISFARR, and R. J. RANDALL: Bio!. Chern. 193, 265 (1951). SMITH, T. A. and G. R. BEST: Phytochem. 16, 841 (1977). STANLEY, R. G. and H. F. LINSKENS: In: Pollen-Biology, Biochemistry, Management, p. 168. Springer-Verlag, Berlin, Heidelberg, New York, 1974. Tupy, ].: In: Genetics To-day, p. 212. Pergamon Press, Oxford, 1963. Tupy, ]., E. HRABETovA, and V. BALATKovA: In: Fertilization in Higher Plants, p. 145. North-Holland Pub!. Co., Amsterdam, 1974. VASIL, I. K. and N. BOSE: Indian. Soc. Bot. Mem. 2, 11 (1959). A. SPERANZA, Institute of Botany, University of Bologna, Bologna, Italy.
Z. Pjlanzenphysiol. Bd. 97. S. 95-102. 1980.
Institute of Botany, University of Bologna, Bologna, Italy
Compounds Released from Incompatible Apple Pollen during in vitro Germination A.
SPERANZA
and G. L.
CALZONI
With 5 figures Received July 16, 1979 . Accepted August 20,1979
Summary Compounds released from pollen tubes of gametophytically controlled apple cultivar were investigated. Pollen was able to regulate the pH of the medium by means of surfacelinked compounds, whose buffering power was evaluated. Proteins, carbohydrates, RNA and polyamines were released in the culture. A possible role of polyamines in fertilization processes is indicated, in support of the hypothesis of extracellular enzyme synthesis controlling pollen tube growth. Key words: pollen germination, pH changes, polyamines, fertilization.
Introduction Pollen of Rosaceae displays a gametophytic monofactorial control of self-incompatibility. A stylar inhibition being involved, it is possible that the pollen incompatibility factors are perhaps synthesized mainly during tube growth rather than pre-stored in the pollen cell-wall (HESLOP-HARRISON, 1977). With this in mind, we searched for some compounds, released during pollen germination of incompatible Malus domestica cultivar, which might be related to the growth regulation of the pollen tube.
Materials and Methods Pollen of Malus domestica (BORKH), cv. Starkrimson, was collected, managed and stored as previously described (CALZONI et al., 1979). Germination tests were performed in 250 ml flasks, constantly shaken (80 cycles/min) for 2 h at 30°C. Liquid medium contained 0.2 M sucrose, 20 ,ug/ml HsB03 and 300 ,ug/ml Ca(NO a)2' 4H20. Different pH values were obtained with 0.01 N HCl or NaOH; in some specified cases citric acid-NaHP0 4 (Mc Ilvai-
Z. Pflanzenphysiol. Bd. 97. S. 95-102. 1980.
96
A. SPERANZA and G. L. CALZONI
ne) buffer was used at 0.01 and 0.01 M respectively. Germination was recorded by photomicrographs after 120 min of incubation; microscopical control in order to check for burst pollen tubes in the culture was negative. Liquid medium was separated from pollen by filtration on Millipore filters (SSWP 04700 SS 3 p,). Filtered medium was stored at -20 DC, in some cases liophylized using an Edwards vacuum pump. Titration of medium was carried out with 0.01 N HCI or NaOH (Normex, Carlo Erba). Proteins released in germination medium were assayed according to LOWRY et ai. (1951). Kirby's method (KIRBY et aI., 1964) modified for plants by LI and ANDERSEN (1967), to extract and determine total RNA was used. Polyamines were detected by direct dansylation according to SMITH and BEST (1977), then dansyl-amines were separated by one-dimensional TLC on Kieselgel 60 plates with concentration zone, 0.25 mm layer thickness (Merck). Plates were run in cyclohexane/ethylacetate (3 : 2), followed immediately by spraying with triethanolamine/isopropanol (1 : 4) to enhance and stabilize the fluorescence. Quantitative analysis of fluorescent compounds was performed in a Jasco spectrofluorometer (excitation and emission wavelengths 365 and 505 nm respectively), and referred to fluorescence of standards run on the same plate. Carbohydrates were assayed in pollen eluate obtained with Coca's solution (COCA, 1922), by a-naphtol test modified according to KIRBY and SMITH (1974).
Results We thorougly re-examined our earlier observations on the ability of pollen to influence the external medium, in particular the pH (CALZONI et aI., 1979). 100 ;;!
z
Q ao
~ ~
0::
~
IGO
•
Buffered medium
.&
Unbuffered mpdium
z
Q40
~ 0:: ~
J:
1"20 I I
0
4
5
6 7 8 INITIAL pH Fig. 1: Per cent pH variations of culture medium and germination of Starkrimson pollen (1 mg/ml), after 120 min incubation at different initial pH, with and without buffer.
z. PJlanzenphysiol. Bd. 97. S. 95-102. 1980.
Compounds released from apple pollen
97
Fig. 1 shows the per cent of pH variation in buffered and unbuffered media; in both cases minimal variations and optimum pH for germination are strictly related; the highest pH changes are associated with the extreme pH values. Therefore, the pollen seems to exert a remarkable control over pH, because all the initial pH values of media become close and similar each other at pH range of 6.0-6.5. It is important to remark that the buffer could not keep the initial pH quite constant, but a low molarity was needed to avoid inhibitory ion effects. Fig. 2 shows that pH changes actually depend on pollen concentration. We may suppose that this pH control is probably due to an elution of substances from pollen cell-walls, rather than to a real activity of living pollen. This elution may be more or less high owing to the pH of medium . 00 f-
8OfI. B uttf'r~
60
mt"cilum
•
ot v.obl," pollf'n
4C f-
2Cf-
0
01
0.5
~
1 2 1" 1" POLLEN mg/ml
Fig. 2 : Per cent pH variations and germination related min of incubation at initial pH 4.
to
pollen concentration, after 120
Really, these pH variations recovered after 120 min of germination are almost established when pollen is suspended in the culture medium, with a gentle shaking of a few seconds. Namely, the highest pH changes occur at this time we have called «zero», since foregoing the real time of incubation at 30 Table 1 shows an attempt to evaluate this buffering power of filtered medium, referred to pure medium at three different pH. The buffering power is most probably due to various ionizable groups of protein-like compounds, easily elutable from pollen cell-walls.
ac.
Z. P/lanzenphysiol. Bd. 97. S. 95-102.1980.
98
A. SPERANZA and G. L. CALZON!
Table 1: Buffer power of germination medium (filtrate) of Starkrimson pollen. A change of one pH unit was considered. Pure medium, taken as reference, was titrated in the same pH interval as filtrate. (For «0» time of germination, see text.) Initial pH
Sample Filtrate Filtrate Pure medium Filtrate Filtrate Pure medium Filtrate Filtrate Pure medium
4.0 4.0 4.0 6.0 6.0 6.0 8.0 8.0 8.0
Germination (min)
Final pH
0 120
6.0 6.1
0 120
6.2 6.3
0 120
6.5 6.5
HCI
NaOH
(.uUml)
(.uUml)
4.5 11.2 3.2 7.5 6.2 5.7 13.7 12.5 8.5
4.5 16.8 3.5 7.0 17.8 4.4 9.4 16.5 6.5
00 0.7
Z40~~"-----:::!350 Z'~:W~2:10'5~--350=' zid Z75
31ia zao
275
~nm
00
b' :--:Z;!;:75~--'3:::!1ia Z~~0:-:;Z'=75"---""3"::!50 Zior.........Z;!;:75,.------=!J50 nm ~""zj;;75"-----:::!350 Z40 00
o
30
60
120 Minutes
Fig. 3: Levels of UV-absorbing materials released by Starkrimson pollen (1 mg/ml) germinating at different initial pH.
Z. Pjlanzenphysiol. Bd. 97. S. 95-102. 1980.
Compounds released from apple pollen
99
.. pH 4
200
• pH 6 • pH 8
:€150 C'I
;z,
Z W I-
0
!fl00
120
Fig. 4: Proteins released in culture medium at different initial pH by germinating pollen (1 mg/ml) of Starkrimson. Levels of UV -absorbing materials released during germination appear to gradually increase (Fig. 3), showing a more and more clear-cut maximum in the general region of proteins (270-280 nm). Nevertheless, a more appropriate assay (LOWRY et al., 1951) was carried out to determine the amounts of released proteins during germination (Fig. 4). Coca's solution (COCA, 1922) was employed to make evident carbohydrates in pollen eluate of our cultivar, so we have a very strong evidence for their presence with a-naphtol test (KIRBY et al., 1974). Moreover, on the basis of Tupf's studies on Nicotiana (Tupf et al., 1974), we tried to identify macromolecules as ribonucleic acids in filtered medium after pollen germination. Small amounts of RNA are present in the liquid medium: 1.4 ,ug/ml in media at both initial pH 4 and 6, and 3.4 ,ug/ml at pH 8. So far these data are indicative of total RNA, without any fractionation. Fig. 5 shows the recovery in pollen filtrate (initial pH 6) of other molecules of great biological importance, like polyamines. They appear to be present at the first stages of germination, then gradually increase, except spermine that shows a very large increment between 60 and 120 min. Moreover, a further interesting issue is observed in TLC plates, where strongly fluorescent compounds with a very low Ram are present. According to DION and HERBST (1970) they are interpretable as
z. PJlanzenphysiol. Bd. 97. S. 95-102. 1980.
100
A. SPERANZA and G. L. CALZONI 4r---------------~-.
.. Sm
:€(5
•
Pu
•
Sd
E3 C
if)
W Z
~
~2
0
CL
o
30
60
MINUTES
120
Fig. 5: Polyamines released by germinating pollen (2 mg/ ml) of Starkrimson, at initial pH 6.
acetyl-polyamines, although quantitative analysis was not made for unavailability of corresponding standards. Acetyl-polyamines are believed to playa role in the control of RNA synthesis (BACHRACH, 1973). Discussion Our data on Malus domestica pollen point to a strong buffering power of its surface-linked compounds. Such control of external pH may also be important in vivo, because it should support pollen germination even when not neutral treatments are carried out in blossoming. Opinions conflict as to whether pH influences pollen germination (VASIL and BOSE, 1959); our data may indicate (Fig. 1) a clear relationship between pH and pollen germination if only a buffer system is present. On the other hand, without any buffer system, germination actually seems to be almost independent of initial pH in the culture medium. An other interesting point is the carbohydrate recovery in pollen eluate of Starkrimson; in this respect, we hypothesize their possible linkage with proteins. In fact, the association of proteins with carbohydrates in pollen cell-wall would
Z. P/lanzenphysiol. Ed. 97. S. 95-102. 1980.
Compounds released from apple pollen
101
facilitate their extraction as glycoproteins (STANLEY and LINSKENS, 1974). According to LEWIS model of gametophytic self-incompatibility (1960), the inhibition factors arise from complexed glycoproteins on the surface of pollen tubes. Concerning the released ribonucleic acid, it is important to remark that RNA could be necessary for an extracellular protein synthesis as supposed by Tupf et al. (1974). In Nicotiana pollen culture (Tupf et al., 1974), fractionation on MAK column indicates rRNA and tRNA in the medium; in addition, the rapid movement of amino acids from pollen tubes (LINSKENS and SCHRAUWEN, 1969) and the occurrence of a new protein in the medium (Tupf, 1963) suggest this possibility. Molar ratio spermidine/spermine is very low if rapported to the inside content of ungerminated pollen of Starkrimson (BAGNI et al., 1978) (SD/SM = 35.5). This fact probably reflects a large polyamine synthesis in growing pollen tubes; this problem is still under ir_vestigation. Polyamines are known to promote cell division in plant and animal tissues. They playa role in almost every step of RNA and protein synthesis; owing to their basic groups they are able to bind with acidic groups, expecially of nucleic acids (BAGNI and SERAFINI-FRACASSINI, 1974). Polyamine recovery in pollen culture medium may suggest a possible role in the pro gamic phase of fertilization processes. Namely, pollen growth in incompatible and compatible systems may depend on released compounds interacting with the stylar tissues. To this purpose, the hypothesis of extracellular protein synthesis may be of great importance, so giving a real significance to polyamines released from pollen tubes. Acknowledgements This work was supported by the Consiglio Nazionale delle Ricerche, «Progetto Finalizzato Biologia della Riproduzione». The authors wish to thank Prof. N. BAGNI for critical reading of the manuscript; they are also indebted with the Defendi Farm for supplying tree branches, and with Dr. G. CIAFARDINI for lyophilizing some materials.
References BACHRACH, U.: In: Function of Naturally Occurring Polyamines, p. 106. Acad. Press, New York and London, 1973. BAGNI, N. and D. SERAFINI FRACASSINI: In: Plant Growth Substances, p. 1205. Hirokawa Pub!. Co. Inc., Tokyo, 1974. BAGNI, N., D. SERAFINI FRACASSINI, V. R. VILLANUEVA, and R. C. ADLAKA: Riv. Ortoflorofrutt. Ita!. 62, 470 (1978). CALZONI, G. L., A. SPERANZA, and N. BAGNI: Scientia Hort. 10,49 (1979). COCA, A. F.: J. Immuno!. 7, 163 (1922). DION, A. S. and E. J. HERBST: Ann. New York Acad. Sci. 171, 723 (1970). HESLOP-HARRISON, J.: In: Incompatibility in Angiosperms, p. 82. Springer-Verlag, Berlin, Heidelberg, New York, 1977.
Z. Pjlanzenphysiol. Bd. 97. S. 95-102. 1980.
102
A. SPERANZA and G. L. CALZONI
KIRBY, E. G. and J. E. SMITH: In: Fertilization in Higher Plants, p. 127. North-Holland Pub!. Co., Amsterdam, 1974. KIRBY, K. S.: In: Progress in Nucleic Acid Research, p. 2. Acad. Press, New York and London, 1964. LEWIS, D.: Proc. Roy. Soc. London, Ser. B, 151, 468 (1960). LI, P. H. and W. R. ANDERSEN: Nature 214,86 (1967). LINSKENS, H. F. and J. SCHRAUWEN: Acta Bot. Neerl. 18, 605 (1969). LOWRY, O. H., N. J. ROSEBROUGH, A. LEWISFARR, and R. J. RANDALL: Bio!. Chern. 193, 265 (1951). SMITH, T. A. and G. R. BEST: Phytochem. 16, 841 (1977). STANLEY, R. G. and H. F. LINSKENS: In: Pollen-Biology, Biochemistry, Management, p. 168. Springer-Verlag, Berlin, Heidelberg, New York, 1974. Tupy, ].: In: Genetics To-day, p. 212. Pergamon Press, Oxford, 1963. Tupy, ]., E. HRABETovA, and V. BALATKovA: In: Fertilization in Higher Plants, p. 145. North-Holland Pub!. Co., Amsterdam, 1974. VASIL, I. K. and N. BOSE: Indian. Soc. Bot. Mem. 2, 11 (1959). A. SPERANZA, Institute of Botany, University of Bologna, Bologna, Italy.
Z. Pjlanzenphysiol. Bd. 97. S. 95-102. 1980.