Abstract

Possible involvement of impaired polyamine biosynthesis in the poor performance of tomato pollen (Lycopersicon esculentum Mill.) at high temperatures was investigated. Incubation of pollen at 38°C suppressed the increase of S-adenosylmethionine decarboxylase (SAMDC) activity in germinating pollen with little influence on arginine decarboxylase activity. Consequently, spermidine and spermine content in the pollen did not increase at 38°C, while putrescine content increased at both 25°C and 38°C. High-temperature inhibition of pollen germination was alleviated by the addition of spermidine or spermine but not of putrescine to the germination medium. Cycloheximide inhibited SAMDC activity in parallel with pollen germination at 25°C, whereas actinomycin D had no effect on either of them, indicating that enhanced SAMDC activity is associated with de novo protein synthesis. Incubation of crude enzyme extracts at 40°C for 1 h did not affect SAMDC. In addition, high temperatures did not enhance protease activity in germinating pollen. These results indicate that low activity of SAMDC, probably due to impaired protein synthesis or functional enzyme formation, is a major cause for the poor performance of tomato pollen at high temperatures.

(Received January 17, 2002; Accepted March 22, 2002)

Introduction

The reproductive organs of plants are generally more vulnerable to damage at high temperature than are the vegetative organs (Stevens and Rudich 1978, Sakata et al. 2000). In tomato (Lycopersicon esculentum Mill.), fruit set is impaired when the maximum daily temperature averages above 32°C or the maximum night temperature averages above 21°C (Moore and Thomas 1952). Recent evidence shows that the primary parameter affecting the fruit set is the mean daily temperature, with day or night temperatures having a secondary role (Peet et al. 1997). Flowering phases most sensitive to high temperatures are meiosis (8–9 d before anthesis) and fertilization (1–3 d after anthesis) (Iwahori and Takahashi 1964, Rudich et al. 1977). Heat stress on pollen mother cells during meiosis may lead to a reduction in pollen fertility, and during fertilization to a reduction in germination and tube growth of viable pollen grains (Dane et al. 1991). It is known that heat sensitivity of pollen differs within tomato cultivars (Rudich et al. 1977, Stevens and Rudich 1978, Hanna and Hernandez 1982, Kalloo 1991). To date, however, little is known about the endogenous factors responsible for such genotypic variations in pollen germinability at high temperatures. Elucidation of these factors may contribute to the breeding strategies for improving crop productivity of heat-sensitive tomato genotypes under high temperature conditions.

Polyamines are polybasic aliphatic amines that are ubiquitous in procaryotic and eucaryotic organisms. It is believed that polyamines play important roles in growth, in key developmental processes of plants and in defense against environmental stresses (Galston and Kaur-Sawhney 1995, Kumar et al. 1997, Martin-Tanguy 1997, Bouchereau et al. 1999). In plants, the diamine putrescine (Put) is synthesized via two main pathways, i.e. ornithine decarboxylase (EC 4.11.17, ODC) and arginine decarboxylase (EC 4.11.19, ADC) pathways using l-Orn and l-Arg as the substrate, respectively (Slocum 1991). The synthesis of the triamine spermidine (Spd) and the tetraamine spermine (Spm) is catalyzed by spermidine synthase and spermine synthase via the incorporation of aminopropyl moiety to Put and to Spd, respectively. S-adenosylmethionine decarboxylase (EC 4.1.1.50, SAMDC) catalyzes the conversion of S-adenosylmethionine to decarboxylated S-adenosylmethionine, a donor of aminopropyl moiety to Put and Spd. The synthesis of Spd and Spm is mainly regulated at the level of SAMDC (Greenburg and Cohen 1985, Tassoni et al. 2000).

Pollen contains a large amount of polyamines as well as high activities of enzymes mediating their biosynthesis. Chibi et al. (Chibi et al. 1993, Chibi et al. 1994) found that isolated tobacco pollen showed the highest ADC activity at the mid-binucleate stage during in vitro incubation at 25°C. Thereafter, ADC activity declined to a very low level towards the maturation stage, and rose again during the early phase of pollen germination. Recently, we found that tomato pollen displayed a marked increase of SAMDC activity in addition to ADC activity within 30 min of incubation at 25°C (Song et al. 2001). Inhibiting SAMDC activity with a SAMDC inhibitor, methylglyoxal bis-(guanylhydrazone) (MGBG), resulted in a substantial decrease of Spd and Spm content in the pollen, and also a great decrease in pollen germination and tube growth. This inhibitory effect of MGBG on pollen germination was completely reversed by the concomitant treatment with Spd or Spm. Put was ineffective in reversing the inhibition caused by MGBG or d-Arg, a competitive inhibitor of ADC. These results show that increased activity of SAMDC and the consequential rise of endogenous Spd and Spm content are essential for pollen germination and tube growth to occur.

Previously, we found that Spd or Spm added to the germination medium or supplied to flowers before anthesis reversed the high-temperature inhibition of in vitro germination and tube growth of tomato pollen (Song et al. 1999). This, together with aforementioned findings, suggests that high-temperature inhibition of pollen germination is mediated by impaired activity of SAMDC resulting in the deficiency of Spd and Spm necessary for pollen germination and tube growth. However, there is little information available about the effect of high temperatures on polyamine biosynthetic enzyme activities and endogenous polyamine levels in germinating pollen of any plant species. Thus, the aim of this study was to elucidate the effect of high incubation temperature on polyamine biosynthesis in germinating pollen and its possible involvement in the poor performance of tomato pollen at elevated temperatures.

Results

Effect of high temperatures on pollen germination and tube growth

The effect of incubation temperatures (25, 34, 36, and 38°C) on germination and tube growth of tomato pollen was investigated. Pollen germination initiated about 20 min after incubation at all temperatures examined. Initially, the inhibitory effect of high temperatures on pollen germination was not large, but pollen grains incubated at higher temperatures ceased germinating sooner. Consequently, the germination percentage was significantly lowered as the temperature was increased above 34°C (Fig. 1A). Tube growth was also slower when the temperature was higher than 34°C, and at 38°C tubes ceased elongating after about 2 h (Fig. 1B).

Effect of high temperatures on polyamine biosynthetic enzyme activities in germinating pollen

Changes in activities of ADC and SAMDC in germinating pollen during the initial 4 h of incubation at 25 and 38°C were investigated. ODC activity was not determined because tomato pollen has negligible ODC activity (Song et al. 2001). Consistent with the previous results (Song et al. 2001), activities of both ADC and SAMDC increased substantially soon after incubation at 25°C (Fig. 2). ADC activity reached a peak after 2 h and remained relatively high thereafter. However, SAMDC activity increased transiently with a peak after 1 h of incubation. It should be noted that activities of both enzymes had already increased 30 min after the start of incubation when the bulk of pollen grains initiated tube emergence. Incubation at 38°C did not affect ADC activity, whereas it completely negated the rise of SAMDC activity. An additional experiment showed that the increase of SAMDC activity was suppressed remarkably at 34°C (Fig. 2).

Effect of high temperatures on polyamine content in germinating pollen

The time course changes of polyamine content in germinating pollen during incubation at 25 and 38°C were determined. At 25°C, free Put content did not increase during the first 30 min, but afterwards it increased steadily until 2 h and remained high thereafter (Fig. 3). Free Spd and Spm content increased transiently but markedly during the first hour of incubation. In the pollen incubated at 38°C, however, Spd and Spm content did not increase throughout the 4-h incubation period, whereas Put content increased similarly to that at 25°C. Consequently, the ratio of Spd plus Spm to Put constantly decreased at 38°C while it increased substantially during the first h of incubation at 25°C.

Conjugated polyamine content decreased substantially during the first hour of incubation at both 25°C and 38°C in all of the three polyamines (Fig. 4). At 25°C, however, they tended to increase after 1 h, and after reaching a peak at 2 h they declined again gradually. Raising the incubation temperature to 38°C exerted little influence on the content of conjugated Put but totally suppressed the increase of conjugated Spd and Spm content. Pollen incubated at 25°C displayed a transient increase in the content of bound Put and bound Spd and Spm after 0.5 and 1 h, respectively. These increases were not observed in pollen incubated at 38°C (data not shown).

Effect of exogenous polyamines on pollen germination and tube growth at high temperatures

The effects of Put, Spd or Spm application to the germination medium on pollen germination and tube growth at 38°C were investigated. The concentration of these polyamines was 0.5 mM based on the results from a preliminary experiment. When the polyamines were applied before incubation, Spd and Spm reversed the inhibitory effects of high temperatures on both pollen germination and tube growth, although incompletely (Fig. 5A). Combining 0.5 mM Spd and 0.1 to 0.5 mM Spm gave no additive effects (data not shown). However, Put and 1,3-diaminopropane, an oxidation product of Spd and Spm, were ineffective, indicating that the effect of Spd and Spm is ascribed to their own function. When the polyamines were applied 2 h after incubation at 38°C, Spd and Spm were also effective in promoting tube growth but without effect on pollen germination (Fig. 5B). Spd did not affect pollen germination or tube growth at 25°C (data not shown).

Effects of RNA and protein synthesis inhibitor on SAMDC activity and pollen germination at normal temperature

The above results indicate that the inability of pollen to enhance SAMDC activity in the early phase of germination is primarily responsible for the poor performance of the pollen at high temperatures. This lack of SAMDC activity increase can arise from impairment of either the transcriptional or the post-transcriptional processes. It is generally accepted that most mRNAs required for pollen germination and early tube growth are already present in the pollen grains at anther dehiscence and remain untranslated until rehydration (Mascarenhas 1993, Taylor and Hepler 1997, Franklin-Tong 1999). However, nothing is known about the features of SAMDC. Therefore, the effects of RNA and protein synthesis inhibitors, actinomycin D and cycloheximide, respectively, on SAMDC activity and pollen germination at 25°C were examined. The inhibitors were applied to the germination medium before incubation. The results showed that actinomycin D did not affect pollen germination or SAMDC activity, whereas cycloheximide severely inhibited both pollen germination and SAMDC activity (Table 1). When the concentration of cycloheximide was very low (0.001–0.01 µg ml–1), it inhibited pollen germination and SAMDC activity to a similar extent (Fig. 6). In addition, the inhibitory effect of 0.005 µg ml–1 cycloheximide on pollen germination was largely overcome by combined treatment with 0.5 mM Spd and 0.25 mM Spm (Fig. 7). These results suggest that SAMDC mRNA is abundantly present in mature pollen while enhanced synthesis of SAMDC proteins takes place in the early phase of pollen germination and its inhibition results in decreased pollen germination.

Thermal stability of SAMDC

To examine the thermal stability of SAMDC in tomato pollen, crude enzyme extract was prepared from pollen incubated at 25°C for 1 h. Then, the enzyme extract was incubated at 30, 35, 40 or 45°C for 1 h before the SAMDC activity was determined at 30°C. SAMDC partially lost its initial activity during incubation at 45°C (Fig. 8); however, incubation at and below 40°C did not decrease the enzyme activity.

Effect of incubation temperature on protease activity in germinating pollen

To test the possibility that SAMDC suffers proteolytic degradation at high temperatures, protease activity in germinating tomato pollen was measured after incubation at 25, 36 or 38°C for 1 h. The result showed that high incubation temperature did not affect protease activity in germinating pollen (Fig. 9).

Discussion

The present results showed that polyamine biosynthesis in germinating tomato pollen was highly susceptible to high temperatures. The time-course changes in polyamine content of pollen incubated at 38°C were similar to those of pollen incubated at 25°C on the medium containing a SAMDC inhibitor MGBG (Song et al. 2001). Therefore, the inability of tomato pollen to increase SAMDC activity at high temperatures (Fig. 2) probably is the main cause for the low content of Spd and Spm in the pollen incubated at 38°C (Fig. 3, 4).

ADC activity was not affected by high temperatures (Fig. 2). However, this does not necessarily indicate that Put biosynthesis was normal at 38°C, because the low activity of SAMDC at 38°C did not result in substantial increases of Put content over that at 25°C (Fig. 3, 4). The synthesis of Put from l-Arg requires the activity of agmatine iminohydrolase (EC 3.4.3.12) and N-carbamoylputrescine amidohydrolase (EC 3.5.1.-) in addition to ADC activity (Slocum 1991). Further study is needed on the high-temperature sensitivity of agmatine iminohydrolase and N-carbamoylputrescine amidohydrolase to fully understand the effect of high temperatures on polyamine biosynthesis in germinating tomato pollen.

High-temperature inhibition of pollen germination and tube growth was alleviated by exogenous Spd and Spm applied to the germination medium before incubation (Fig. 5A). This result is consistent with the finding by Balasundaram et al. (1996), who found that a polyamine-deficient mutant of Saccharomyces cerevisiae was very sensitive to high temperatures and its severe growth inhibition at 39°C was mitigated by 0.1 mM Spd applied to the growth medium. More recently, Hanzawa et al. (2000) found that an Arabidopsis mutant with a stunted phenotype restores normal stem elongation by the introduction of the Arabidopsis ACAULIS5 gene that encodes spermine synthase. Spd and Spm failed to promote germination of tomato pollen at 38°C, when applied to the germination medium 2 h after incubation, although pollen tube growth was significantly promoted (Fig. 5B). In addition, the increase of SAMDC activity at 25°C was significant when the bulk of pollen grains initiated tube emergence (Fig. 1, 2). Thus, it is inferred that tomato pollen requires the early enhancement of SAMDC activity with a concomitant increase of free Spd and Spm content for tube emergence. Previously, we found that Spd and Spm completely reversed the MGBG-induced inhibition of pollen germination and tube growth (Song et al. 2001). However, the effect of these polyamines in reversing the adverse effect of high temperatures was incomplete, despite the similar impact on polyamine content in germinating pollen. This indicates that high temperatures may have impaired some essential processes independent of SAMDC activity. Taken together, it is concluded that inability of pollen to enhance SAMDC activity is a major but not the sole cause for the poor performance of tomato pollen at high temperatures.

Several experiments were performed to clarify the cause for the lack of SAMDC activity increase in tomato pollen incubated at high temperatures. Actinomycin D did not affect either SAMDC activity or pollen germination at 25°C while cycloheximide severely inhibited both of them (Table 1). The results are consistent with a recent report by Fernando et al. (2001) and show that sufficient amounts of SAMDC mRNA are presynthesized during pollen maturation and their enhanced translation takes place during the early phase of pollen germination. Thus, the lack of increased SAMDC activity at high temperatures may well be attributable to the inhibition of functional SAMDC protein synthesis or to the loss of activity of newly synthesized SAMDC due to heat inactivation or proteolytic degradation. Incidentally, the results in Fig. 6 and 7 may suggest that blockage of SAMDC protein synthesis accounts for most of the impaired pollen germination by cycloheximide. Although the results are very interesting, further study is needed as to whether Spd and Spm can interfere with the mode of cycloheximide action.

In subsequent experiments, we investigated the thermal stability of SAMDC and the effect of incubation temperature on protease activity in germinating pollen. Although the thermal stability of SAMDC in higher plants has not yet been studied, Cacciapuoti et al. (1991) found that SAMDC purified from an extreme thermophilic archaebacterium Sulfolobus solfataricus did not lose activity during incubation at 50°C for 16 h. In tomato pollen, exposure of crude enzyme extracts to 45°C for 1 h resulted in partial loss of SAMDC activity (Fig. 8). However, incubation at 40°C did not cause loss of enzyme activity. In addition, protease activity in pollen incubated at either 36 or 38°C was not different from that at 25°C (Fig. 9), indicating that the high temperatures did not stimulate the synthesis of protease in germinating pollen. Thus, the possibility that SAMDC newly synthesized in germinating pollen is rapidly heat-inactivated or proteolyzed during incubation at 38°C can be disregarded.

Overall, it is most probable that the processes associated with the formation of functional SAMDC have been impaired by high temperatures. These processes include SAMDC mRNA translation, processing of the translation product and protein folding. SAMDC is first formed as an inactive proenzyme, and the proenzyme must be cleaved to form the pyruvoyl-containing functional enzyme (Kashiwagi et al. 1990, Dresselhaus et al. 1996). In addition, after the completion of translation, SAMDC must fold into a precise three-dimensional structure to carry out its biological function (Gething and Sambrook 1992). Our results do not allow us to speculate which of the processes was impaired by high temperatures. However, it is interesting to note that high incubation temperature exerted little influence on ADC activity despite the complete suppression of SAMDC activity increase (Fig. 2). ADC is also formed as an inactive proenzyme but the proenzyme is cleaved via a processing enzyme, while SAMDC is auto-catalytic self-processing (Malmberg and Cellino 1994, Borrell et al. 1996, Galston et al. 1997). In addition, ADC does not contain a pyruvate prosthetic group in the molecule (Cohen 1998). Further study is necessary to clarify the mechanism by which functional SAMDC formation is impaired in tomato pollen exposed to high temperatures. From an agricultural viewpoint, whether the genetic variations in pollen germinability at high temperatures involve a different susceptibility to high temperature of the processes associated with the functional SAMDC formation deserves further investigation.

In summary, the results of the present study show clearly that the low activity of SAMDC in germinating pollen is a major cause for the poor performance of tomato pollen at high temperatures. This low activity of SAMDC probably results from impaired enzyme protein synthesis or functional enzyme formation by high temperatures.

Materials and Methods

Pollen collection, storage, and germination in vitro

Pollen grains of tomato (L. esculentum Mill., cv. Jifan No. 3) were collected from flowers freshly opened in the greenhouse during winter and spring seasons with daily maximum air temperatures not exceeding 30°C. The collected pollen grains were dried over silica gel for 24 h prior to storage at –30°C in a desiccator. Incubation of pollen grains, chemical composition of pollen germination medium, counting of germinated pollen grains, and tube length measurements were as described previously (Song et al. 2001). Measurements and assays were repeated at least three times in all of the experiments.

Assay of polyamine biosynthetic enzyme activity

Pollen grains (20 mg) were incubated in a centrifuge tube with shaking. After collecting the pollen by centrifugation, enzymes were extracted in 100 mM potassium phosphate (pH 8.0), 20 mM sodium ascorbate, 1 mM pyridoxal-5′-phosphate, 10 mM dithiothreitol, 0.1 mM EDTA and 0.1 mM phenylmethylsulfonyl fluoride (Chibi et al. 1994). After centrifugation at 25,000×g for 20 min, the supernatant was dialyzed overnight against the extraction buffer. Enzyme activity was determined at 30°C as described by Song et al. (2001). The substrates used for ADC and SAMDC activities were 9 mM l-Arg labeled with 185 kBq ml–1l-[U-14C]Arg and 2.7 mM S-adenosylmethionine labeled with 92.5 kBq ml–1 S-adenosyl-l-[carboxyl-14C]methionine, respectively. Protein concentrations in enzyme extracts were determined by the method of Bradford (1976). Enzyme activity is based on the weight of pollen grains before incubation.

Assay of protease activity

Protease activity was assayed as described by Belles et al. (1991). Pollen grains (20 mg) were incubated in 5 ml of germination medium at different temperatures for 1 h. After pollen collection by centrifugation, protease was extracted in 50 mM phosphate-citrate buffer (pH 6.6) containing 20 mM KCl, 2 mM MgCl2, 5% (w/v) sucrose and 15 mM 2-mercaptoethanol. The homogenate was centrifuged at 30,000×g for 30 min at 2°C. The resulting supernatant was used to determine protease activity.

The reaction mixture (total volume of 150 µl) contained 20 µl of enzyme extract, 120 µl of assay buffer and 10 µl of 0.5% (w/v) FITC-casein (Sigma, U.S.A.). The assay buffer was 150 mM phosphate-citrate buffer (pH 6.6) containing 0.5 mM dithiothreitol. Assay was carried out at 37°C for 1 h, and the reaction was stopped by adding 150 µl of 10% (w/v) trichloroacetic acid. After standing on ice for 1 h, the reaction mixture was centrifuged at 5,000×g for 20 min. The supernatant (200 µl) was mixed with 3 ml of 500 mM Tris-HCl (pH 8.5) and its fluorescence was determined in a fluorescence spectrophotometer with an excitation wavelength of 490 nm and an emission wavelength of 525 nm. One unit of FITC-casein-degrading activity is defined as the enzyme needed to produce one unit of fluorescence increase under the standard assay conditions.

Polyamine analysis

After incubation of pollen grains (10 mg) in a centrifuge tube with shaking, the germination medium was freeze-dried because polyamines could be released into the medium from germinating pollen (Speranza and Calzoni 1980). Polyamines (free, conjugated and bound forms of Put, Spd and Spm) were quantified as described by Song et al. (2001). Briefly, polyamines were extracted in 5% (w/v) perchloric acid. After centrifugation, the supernatant was preserved and the pellet was resuspended in 5% perchloric acid after several washes with the same solution. Aliquots of acid-soluble and acid-insoluble fractions, containing free plus conjugated polyamines and bound polyamines, respectively, were subjected to hydrolysis in 6 M HCl at 110°C for 18 h to convert the conjugated and bound forms to free form. After the hydrolyzate was taken to dryness in an aluminum bath at 70°C, the residues were dissolved in a small amount of 5% perchloric acid. Following dansylation, polyamines in the supernatant and hydrolyzate were quantified by HPLC using a µ-Bondapack C18 reverse phase column (Waters, U.S.A.) with a µ-Bondapack C18 guard column. Conjugated polyamine content was calculated by subtracting free polyamine content from total acid-soluble polyamine content. 1,6-Hexanediamine was used as an internal standard. Polyamine content is based on the weight of pollen grains before incubation.

Treatments of pollen with polyamines and inhibitors of RNA and protein synthesis

Polyamines (di-, tri- and tetra-hydrochloride salts of Put, Spd and Spm, respectively), and actinomycin D and cycloheximide, RNA and protein biosynthesis inhibitors respectively, were added to the germination medium before or during pollen incubation. These treatments did not affect the pH of the medium.

1

Corresponding author: E-mail, tatibana@bio.mie-u.ac.jp; Fax, +81-59-231-9637.

Fig. 1 Time-course changes in germination percentages (A) and tube length (B) of tomato pollen during incubation at different temperatures. Vertical bars indicate SE (n = 3).

Fig. 1 Time-course changes in germination percentages (A) and tube length (B) of tomato pollen during incubation at different temperatures. Vertical bars indicate SE (n = 3).

Fig. 2 Time-course changes in arginine decarboxylase (ADC) and S-adenosylmethionine decarboxylase (SAMDC) activities in tomato pollen during incubation at different temperatures. Enzyme activity is based on the pollen weight before incubation. Vertical bars indicate SE (n = 3).

Fig. 2 Time-course changes in arginine decarboxylase (ADC) and S-adenosylmethionine decarboxylase (SAMDC) activities in tomato pollen during incubation at different temperatures. Enzyme activity is based on the pollen weight before incubation. Vertical bars indicate SE (n = 3).

Fig. 3 Time-course changes in free polyamine content of tomato pollen during incubation at 25 and 38°C. Polyamine content is based on the pollen weight before incubation. Vertical bars indicate SE (n = 3).

Fig. 3 Time-course changes in free polyamine content of tomato pollen during incubation at 25 and 38°C. Polyamine content is based on the pollen weight before incubation. Vertical bars indicate SE (n = 3).

Fig. 4 Time-course changes in conjugated polyamine content of tomato pollen during incubation at 25 and 38°C. Polyamine content is based on the pollen weight before incubation. Vertical bars indicate SE (n = 3).

Fig. 4 Time-course changes in conjugated polyamine content of tomato pollen during incubation at 25 and 38°C. Polyamine content is based on the pollen weight before incubation. Vertical bars indicate SE (n = 3).

Fig. 5 Effect of exogenous polyamines on germination percentages and tube length of tomato pollen incubated at 38°C. Polyamines were added to the germination medium before (A) or 2 h after incubation (B). The data were taken after 4 and 6 h of incubation in A and B, respectively. Dap represents 1,3,-diaminopropane. Means with different letters are significantly different at P<0.05.

Fig. 5 Effect of exogenous polyamines on germination percentages and tube length of tomato pollen incubated at 38°C. Polyamines were added to the germination medium before (A) or 2 h after incubation (B). The data were taken after 4 and 6 h of incubation in A and B, respectively. Dap represents 1,3,-diaminopropane. Means with different letters are significantly different at P<0.05.

Fig. 6 Effect of low concentrations of cycloheximide on S-adenosylmethionine decarboxylase (SAMDC) activity and germination percentages of tomato pollen incubated at 25°C. Enzyme activity and pollen germination percentages were determined after incubation for 1 and 2 h, respectively. Enzyme activity is based on the pollen weight before incubation. Means with different letters are significantly different at P<0.05.

Fig. 6 Effect of low concentrations of cycloheximide on S-adenosylmethionine decarboxylase (SAMDC) activity and germination percentages of tomato pollen incubated at 25°C. Enzyme activity and pollen germination percentages were determined after incubation for 1 and 2 h, respectively. Enzyme activity is based on the pollen weight before incubation. Means with different letters are significantly different at P<0.05.

Fig. 7 Effect of addition of 0.5 mM spermidine with or without combined addition of 0.1 to 0.5 mM spermine to the cycloheximide-containing medium on germination percentages (A) and tube length (B) of tomato pollen after incubation at 25°C for 2 h. The cycloheximide concentration was 0.005 µg ml–1. Means with different letters are significantly different at P<0.05.

Fig. 7 Effect of addition of 0.5 mM spermidine with or without combined addition of 0.1 to 0.5 mM spermine to the cycloheximide-containing medium on germination percentages (A) and tube length (B) of tomato pollen after incubation at 25°C for 2 h. The cycloheximide concentration was 0.005 µg ml–1. Means with different letters are significantly different at P<0.05.

Fig. 8 Thermal stability of S-adenosylmethionine decarboxylase in tomato pollen. Enzymes were extracted from pollen after incubation at 25°C for 1 h. The crude enzyme extract was incubated at the indicated temperatures for 1 h before the enzyme activity was assayed. The enzyme activity before incubation (control) was 0.56 nmol (mg protein) –1 h–1. Means with different letters are significantly different at P<0.05.

Fig. 8 Thermal stability of S-adenosylmethionine decarboxylase in tomato pollen. Enzymes were extracted from pollen after incubation at 25°C for 1 h. The crude enzyme extract was incubated at the indicated temperatures for 1 h before the enzyme activity was assayed. The enzyme activity before incubation (control) was 0.56 nmol (mg protein) –1 h–1. Means with different letters are significantly different at P<0.05.

Fig. 9 Effect of incubation temperatures on protease activity in germinating tomato pollen. Pollen grains were incubated at 25, 36 and 38°C for 1 h before the enzyme activity was assayed at 37°C. Enzyme activity is based on the pollen weight before incubation. Means with the same letters are not significantly different at P<0.05.

Fig. 9 Effect of incubation temperatures on protease activity in germinating tomato pollen. Pollen grains were incubated at 25, 36 and 38°C for 1 h before the enzyme activity was assayed at 37°C. Enzyme activity is based on the pollen weight before incubation. Means with the same letters are not significantly different at P<0.05.

Table 1

Effect of actinomycin D and cycloheximide on germination percentages, tube length and SAMDC activity in tomato pollen

Inhibitors Conc. (µg ml–1% Germination  Tube length (µm)  SAMDC activity (pmol (mg FW)–1 h–1
  2 h  2 h 4 h  1 h 
Control – 59.2 a  308.2 a 701.3 a  26.2 a 
Actinomycin D 10 60.1 a  304.8 a 708.5 a  ND 
 25 58.9 a  312.6 a 696.1 a  ND 
 50 59.6 a  306.1 a 698.3 a  24.9 a 
Cycloheximide 10 5.7 b  57.5 b 58.3 b  ND 
 20 4.3 b  52.6 b 52.6 b  ND 
 30 0.0 c  0.0 c 0.0 c  3.8 b 
Inhibitors Conc. (µg ml–1% Germination  Tube length (µm)  SAMDC activity (pmol (mg FW)–1 h–1
  2 h  2 h 4 h  1 h 
Control – 59.2 a  308.2 a 701.3 a  26.2 a 
Actinomycin D 10 60.1 a  304.8 a 708.5 a  ND 
 25 58.9 a  312.6 a 696.1 a  ND 
 50 59.6 a  306.1 a 698.3 a  24.9 a 
Cycloheximide 10 5.7 b  57.5 b 58.3 b  ND 
 20 4.3 b  52.6 b 52.6 b  ND 
 30 0.0 c  0.0 c 0.0 c  3.8 b 

Incubation was carried out at 25°C for up to 4 h.

Means with different letters are significantly different at P<0.05.

ND represents not determined.

References

Balasundaram, D., Tabor, C.W. and Tabor, H. (
1996
) Sensitivity of polyamine-deficient Saccharomyces cerevisiae to elevated temperatures.
J. Bacteriol.
 
178
:
2721
–2724.
Belles, J.M., Carbonell, J. and Conejero, V. (
1991
) Polyamines in plants infected by citrus exocortis viroid or treated with silver ions and ethephon.
Plant Physiol.
 
96
:
1053
–1059.
Borrell, A., Besford, R.T., Altabella, T., Masgrau, C. and Tiburcio, A.F. (
1996
) Regulation of arginine decarboxylase by spermine in osmotically-stressed oat leaves.
Physiol. Plant.
 
98
:
105
–110.
Bouchereau, A., Aziz, A., Larher, F. and Martin-Tanguy, J. (
1999
) Polyamines and environmental challenges.
Plant Sci.
 
140
:
103
–125.
Bradford, M.M. (
1976
) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-die binding.
Anal. Biochem.
 
72
:
248
–254.
Cacciapuoti, G., Porcelli, M., De Rosa, M., Gambacorta, A. and Bertoldo, C. (
1991
) S-adenosylmethionine decarboxylase from the thermophilic archaebacterium Sulfobus solfataricus. Purification, molecular properties and studies on the covalently bound pyruvate.
Eur. J. Biochem.
 
199
:
395
–400.
Chibi, F., Angosto, T., Garrido, D. and Matilla, A. (
1993
) Requirement of polyamines for in vitro maturation of the mid-binucleate pollen of Nicotiana tabacum.
J. Plant Physiol.
 
142
:
452
–456.
Chibi, F., Matilla, A.J. and Garrido, D. (
1994
) Changes in polyamine synthesis during anther development and pollen germination in tobacco (Nicotiana tabacum).
Physiol. Plant.
 
92
:
61
–68.
Cohen, S.S. (
1998
) A Guide to the Polyamines. pp. 595. Oxford University Press, New York.
Dane, F., Hunter, A.G. and Chambliss, O.L. (
1991
) Fruit set, pollen fertility and combining ability of selected tomato genotypes under high-temperature field conditions.
J. Amer. Soc. Hort. Sci.
 
116
:
906
–910.
Dresselhaus, T., Barcelo, P., Hagel, C., Lorz, H. and Humbeck, K. (
1996
) Isolation and characterization of a tritordeum cDNA encoding S-adenosylmethionine decarboxylase that is circadian-clock-regulated.
Plant Mol. Biol.
 
30
:
1021
–1033.
Fernando, D.D., Owens, J.N., Yu, X. and Ekramoddoullah, A.K.M. (
2001
) RNA and protein synthesis during in vitro pollen germination and tube elongation in Pinus monticola and other conifers.
Sex. Plant Reprod.
 
13
:
259
–264.
Franklin-Tong, V.E. (
1999
) Signaling and the modulation of pollen tube growth.
Plant Cell
 
11
:
727
–738.
Galston, A.W. and Kaur-Sawhney, R. (
1995
) Polyamines as endogenous growth regulators. In Plant Hormones: Physiology, Biochemistry and Molecular Biology. Edited by Davies, P.J. pp.
158
–178. Kluwer Academic Publishers, Dordrecht.
Galston, A.W., Kaur-Sawhney, J., Altabella, T. and Tiburcio, A.F. (
1997
) Plant polyamines in reproductive activity and response to abiotic stress.
Bot. Acta
 
110
:
197
–207.
Gething, M.-J. and Sambrook, J. (
1992
) Protein folding in the cell.
Nature
 
355
:
33
–45.
Greenburg, M.L. and Cohen, S.S. (
1985
) Dicyclohexylamine-induced shift of biosynthesis from spermidine to spermine in plant protoplasts.
Plant Physiol.
 
78
:
568
–575.
Hanna, H.Y. and Hernandez, T.P. (
1982
) Response of six tomato genotypes under summer and spring conditions in Louisiana.
HortScience
 
17
:
758
–759.
Hanzawa, Y., Takahashi, T., Michael, A.J., Burtin, D., Long, D., Pineiro, M., Coupland, G. and Komeda, Y. (
2000
) ACAULIS5, an Arabidopsis gene required for stem elongation, encodes a spermine synthase.
EMBO J.
 
19
:
4248
–4256.
Iwahori, S. and Takahashi, K. (
1964
) High temperature injuries in tomato. III. Effects of high temperature on flower buds and flowers of different stages of development.
J. Japan. Soc. Hort. Sci.
 
33
:
67
–74.
Kalloo, G. (
1991
) Breeding for environmental stress resistance in tomato. In Genetic Improvement of Tomato. Edited by Kalloo, G. pp.
153
–165. Springer-Verlag, Berlin.
Kashiwagi, K., Taneja, S.K., Liu, T.-Y., Tabor, C.W. and Tabor, H. (
1990
) Spermidine biosynthesis in Saccharomyces cerevisiae. Biosynthesis and processing of a proenzyme form of S-adenosylmethionine decarboxylase.
J. Biol. Chem.
 
265
:
22321
–22328.
Kumar, A., Altabella, T., Taylor, M.A. and Tiburcio, A.F. (
1997
) Recent advances in polyamine research.
Trends Plant Sci.
 
2
:
124
–130.
Malmberg, R.L. and Cellino, M.L. (
1994
) Arginine decarboxylase of oats is activated by enzymatic cleavage into two polypeptides.
J. Biol. Chem.
 
269
:
2703
–2706.
Martin-Tanguy, J. (
1997
) Conjugated polyamines and reproductive development: biochemical, molecular and physiological approaches.
Physiol. Plant.
 
100
:
675
–688.
Mascarenhas, J.P. (
1993
) Molecular mechanisms of pollen tube growth and differentiation.
Plant Cell
 
5
:
1303
–1314.
Moore, E.L. and Thomas, W.O. (
1952
) Some effects of shading and para-chlorophenoxyacetic acid on fruitfulness of tomato.
Proc. Amer. Soc. Hort. Sci.
 
60
:
289
–294.
Peet, M.M., Willits, D.H. and Gardner, R. (
1997
) Response of ovule development and post-pollen production processes in male-sterile tomatoes to chronic, sub-acute high temperature stress.
J. Exp. Bot.
 
48
:
101
–112.
Rudich, J., Zamski, E. and Regev, Y. (
1977
) Genotypic variation for sensitivity to high temperature in the tomato: pollination and fruit set.
Bot. Gaz.
 
138
:
448
–452.
Sakata, T., Takahashi, H., Nishiyama, I. and Higashitani, A. (
2000
) Effects of high temperature on the development of pollen mother cells and microspores in barley Hordeum vulgare L.
J. Plant Res.
 
113
:
395
–402.
Slocum, R.D. (
1991
) Polyamine biosynthesis in plants. In Biochemistry and Physiology of Polyamines in Plants. Edited by Slocum, R.D. and Flores, H.E. pp.
23
–40. CRC Press, Florida.
Song, J., Nada, K. and Tachibana, S. (
1999
) Ameliorative effect of polyamines on the high temperature inhibition of in vitro pollen germination in tomato (Lycopersicon esculentum Mill.).
Sci. Hortic.
 
80
:
203
–212.
Song, J., Nada, K. and Tachibana, S. (
2001
) The early increase of S-adenosylmethionine decarboxylase activity is essential for the normal germination and tube growth in tomato (Lycopersicon esculentum Mill.) pollen.
Plant Sci.
 
161
:
507
–515.
Speranza, A. and Calzoni, G.L. (
1980
) Compounds released from incompatible apple pollen during in vitro germination.
Z. Pflanzenphysiol.
 
97
:
95
–102.
Stevens, M.A. and Rudich, J. (
1978
) Genetic potential for overcoming physiological limitations on adaptability, yield, and quality in the tomato.
HortScience
 
13
:
673
–678.
Taylor, L.P. and Hepler, P.K. (
1997
) Pollen germination and tube growth.
Annu. Rev. Plant Physiol. Plant Mol. Biol.
 
48
:
461
–491.
Tassoni, A., Van Buuren, M., Franceshetti, M., Fornalé, S. and Bagni, N. (
2000
) Polyamine content and metabolism in Arabidopsis thaliana and effect of spermidine on plant development.
Plant Physiol. Biochem.
 
38
:
383
–393.