JXB Advance Access originally published online on December 7, 2007
Journal of Experimental Botany 2007 58(15-16):4235-4244; doi:10.1093/jxb/erm280
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© 2007 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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RESEARCH PAPER |
Loss of viability of tomato pollen during long-term dry storage is associated with reduced capacity for translating polyamine biosynthetic enzyme genes after rehydration
Laboratory of Horticultural Crop Physiology, Faculty of Bioresources, Mie University, Tsu, Mie 514-8507, Japan
To whom correspondence should be addressed. E-mail: spermidine819{at}yahoo.co.jp
Received 1 August 2007; Revised 12 October 2007 Accepted 15 October 2007
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Abstract |
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The possibility that a loss of pollen viability during dry storage in a freezer is caused by the reduced pollen capacity to enhance polyamine biosynthetic enzyme activity after rehydration was investigated using pollen grains of tomato (Solanum lycopersicum=Lycopersicon esculentum) stored at –30 °C under dry conditions for up to 42 months. Pollen grains showed normal germinability for at least 12 months in storage, but those stored for longer than 24 months exhibited a significant reduction in germinability and fruit-setting ability. This age-dependent reduction in pollen viability coincided with the extent to which the pollen lost the capacity to increase arginine decarboxylase (ADC) and S-adenosylmethionine decarboxylase (SAMDC) activities and polyamine contents upon rehydration. Immunoblot analysis indicated that the capacity of pollen to translate ADC and SAMDC mRNAs was impaired in accordance with the loss of viability. Also, the capacity to synthesize proteins in general decreased with the increase in storage duration. The addition of 1 mM putrescine, spermidine, or spermine to incubation medium promoted germination, impregnation of pollen grains with 1 mM spermidine restored fertilization ability, and the addition of 1 mM spermidine to incubation medium promoted protein synthesis exclusively in pollen grains which had been stored for a long time. These results indicate that the reduction in viability of tomato pollen during long-term dry storage in a freezer involves a decline in the capacity to enhance gene translation for polyamine biosynthetic enzymes upon rehydration.
Key words: S-Adenosylmethionine decarboxylase, arginine decarboxylase, pollen longevity, pollen storage, polyamine, protein synthesis, tomato (Solanum lycopersicum)
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Introduction |
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Pollen storage is required for breeding programmes and genetic conservation, and artificial pollination of dichogamous, self-incompatible, or male-sterile flowers. In such cases, pollen grains have to be stored for extended periods of time without a significant loss of viability. Longevity of pollen, defined as the period of time over which the pollen retains its viability, i.e. germinability and fertilization ability, varies greatly with plant species and storage conditions (Hanna and Towill, 1995; Dafni and Firmage, 2000). The pollen of gramineous plants such as rice, wheat, and maize is extremely short-lived with a life span from several minutes to hours (Luna et al., 2001). By contrast, cattail pollen has a maximum longevity of about 120 d at 24 °C when the pollen grains are placed in an atmosphere of 40% relative humidity (van Bilsen and Hoekstra, 1993).
One of major causes for such variations in pollen longevity among plant species has been attributed to the difference in desiccation tolerance of the pollen. Pollen grains of the family Gramineae are generally unable to survive air-drying and this susceptibility to damage from drying limits their longevity (Hoekstra et al., 1989; Buitink et al., 1996). However, most pollen, especially bicellular pollen including tomato pollen, is desiccation-tolerant and under dry conditions can be stored for extended periods of time without loss of viability (Hanna and Towill, 1995). Pollen grains of tomato stored in open air lose half of their original germination capacity within 2 d at 25 °C and within 5 d at 6 °C (Abdul-Baki, 1992), while those stored at –20 °C under dry conditions retain viability for >3 years (Hanna and Towill, 1995). This storage stability of dry pollen grains has been associated with the occurrence of a glassy state (a highly viscous, semi-equilibrium solid liquid state) in the cytoplasm (Buitink and Leprince, 2004). The high viscosity of intracellular glasses has been considered to decrease molecular mobility and impede diffusion within the cytoplasm, thereby slowing down the deterioration rate of the pollen during storage.
Nevertheless, the longevity of dry desiccation-tolerant pollen is only at a maximum for 1–3 years even if they are stored at –20 °C to –30 °C (Hanna and Towill, 1995), indicating that some deleterious physical and chemical changes proceed gradually in the refrigerator-stored dry pollen. Many studies have indicated that pollen deterioration during ageing involves disrupted intracellular integrity, decreased activity of enzymes like cytochrome oxidase, accumulation of free radicals, and de-esterification and peroxidation of membrane lipids leading to increased leakage of cellular components upon rehydration (imbibitional leakage) (Priestley et al., 1985; Georgieva and Kruleva, 1994; van Bilsen et al., 1994; Taylor and Hepler, 1997). It has been shown that the ageing-mediated membrane damage of pollen is not associated with protein denaturation (Wolkers and Hoekstra, 1995). However, little information is available about whether the age-dependent loss of pollen viability is associated with the reduced capacity of pollen to synthesize proteins after rehydration, an imperative factor for the onset of tube emergence in bicellular pollen (Hoekstra and Bruinsma, 1979; Mascarenhas, 1993; Hiscock et al., 1995). Thus, the precise cause of pollen viability loss is still not well defined.
Polyamines such as a diamine putrescine (Put), a triamine spermidine (Spd), and a tetra-amine spermine (Spm) are aliphatic polycations present in almost all living cells. In plants, the synthesis of Put is catalysed by ornithine decarboxylase (EC 4.1.1.1 [EC] 7) and arginine decarboxylase (EC 4.1.1.1 [EC] 9, ADC) with L-ornithine and L-arginine as substrates, respectively (Bagni and Tassoni, 2001). Put is converted to Spd and further to Spm by the addition of aminopropyl moiety to one and both amino groups of Put, respectively. The aminopropyl donor is decarboxylated S-adenosylmethionine (SAM), which is derived from SAM in a reaction catalysed by SAM decarboxylase (EC 4.1.1.5 [EC] 0, SAMDC). Polyamines occur in cells not only in the free form but also in the acid-soluble conjugated form and the acid-insoluble bound form. Conjugated polyamines are most commonly linked to hydroxycinnamic acid monomers, while bound polyamines are linked to hydroxycinnamic acid dimers and trimers and macromolecules like nucleic acids and proteins.
Polyamines have been shown to play important roles in pollen germination and tube growth (Bagni et al., 1981; Prakash et al., 1988; Galston and Sawhney, 1990; Chibi et al., 1994, Bais and Ravishankar, 2002). It had previously been found that tomato pollen displayed a marked increase in ADC and SAMDC activities and free polyamine contents after 30 min of incubation in liquid medium at 25 °C (Song et al., 2001). Ornithine decarboxylase activity was undetectable throughout the incubation period (4 h), and thus ADC serves as the only enzyme catalysing the synthesis of Put in tomato pollen. Impaired germination and tube growth of tomato pollen at elevated temperatures (e.g. 38 °C) was associated with the failure of SAMDC activity and Spd and Spm contents to rise—the addition of Spd or Spm to germination medium alleviated heat-induced inhibition of pollen germination (Song et al., 2002). These results demonstrate that endogenous levels of Spd and/or Spm play a pivotal role in germination and tube elongation of tomato pollen. Thus, it seems possible that the loss of pollen viability during long-term dry storage in a freezer might involve a reduction in the capacity of pollen to enhance the activity of polyamine biosynthetic enzymes upon rehydration. The present study was undertaken to examine this hypothesis using tomato pollen grains stored at –30 °C under dry conditions for up to 42 months.
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Materials and methods |
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Source of pollen
Tomato plants (Solanum lycopersicum L.=Lycopersicon esculentum Mill., cv. Jifan No. 3) were grown in clay pots filled with commercial nursery soil under greenhouse conditions in the spring or autumn of the years 2000–2004. Anthers were collected from flowers at anthesis and dried over silica gel in a Petri dish at room temperature for 1 d to facilitate dehiscence. Pollen grains were sampled by pressing the anthers with forceps and stored at –30 °C in a tightly sealed vial containing several pieces of dry silica gel. Such procedures were repeated every day for about 10 d until sufficient amounts of pollen grains were collected for a season. Then, all of the pollen grains collected in a season were thoroughly mixed for uniformity, after which a portion of the pollen grains was sampled to assay their germinability and the remainder were again stored in the freezer. The following assays were conducted one by one in the spring of 2004 using the pollen grains stored for 1, 12, 24, 36, and 42 months. For the 1-month-old pollen grains, assays were initiated 1 d after mixing pollen grains collected every day for about 10 d in the spring of 2004. Since it took about 20 d to complete these assays, the exact ages of 1-month-old pollen grains when an assay was executed ranged from about 2 d to 30 d after release from anthers.
Assessment of pollen viability
Pollen viability was assessed in terms of in vitro germinability and fertilization ability of the pollen. For assaying germinability, pollen grains were incubated at 25 °C in a 30 µl liquid germination medium mounted on a slide glass. The medium consisted of 20 mM MES (pH 6.0), 2% (w/v) sucrose, 15% (w/v) polyethylene glycol 4000, 1 mM KNO3, 3 mM Ca(NO3)2, 0.8 mM MgSO4, and 1.6 mM H3BO3. In some experiments, the germination medium was supplemented with polyamines (Put, Spd, or Spm at 1 mM) and a protein synthesis inhibitor (cycloheximide at 10 ng ml–1). Prior to being placed on the medium, pollen grains were hydrated for 1 h at room temperature in an atmosphere of 95% RH that was produced by saturated Na2HPO4·12H2O solution (Winston and Bates, 1960; Sato et al., 1998). This prehydration procedure is necessary to prevent imbibitional damage to the membranes from occurring (Shivanna and Heslop-Harrison, 1981). After 2 h of incubation, pollen germination percentage and tube length were determined as described previously (Song et al., 2001).
The fertilization ability of pollen was assessed in terms of percentage fruit set on emasculated flowers following pollination with pollen grains under investigation. In this experiment, a batch of pollen grains was impregnated with Spd before pollination. For this purpose, pollen grains were soaked in 1 mM Spd solution for 5 min at room temperature, after which they were collected on filter paper, dried in a desiccator for 1 d, and stored at –30 °C in a sealed vial until being used for pollination experiments. Four emasculated flowers on the first inflorescence of greenhouse-grown tomato plants at anthesis (six plants and thus 24 flowers per treatment) were each hand-pollinated with Spd-impregnated or control pollen grains which had been stored for 1, 24, and 42 months. The number of fruits set was counted 20 d after pollination.
Assay of polyamine biosynthetic enzyme activity and polyamine content
The activity of ADC and SAMDC and the content of acid-soluble free and conjugated polyamines and acid-insoluble bound polyamines in pollen grains were determined before and after incubation for 60 min as described previously (Song et al., 2001). Ornithine decarboxylase activity was not assayed because its activity is negligible in mature tomato pollen grains (Song et al., 2001). The method for pollen incubation here and in the molecular analysis described below was as follows. Pollen grains (10 mg) were prehydrated as above, transferred to a 50 ml plastic tube containing 5 ml of temperature-preconditioned medium, and incubated at 25 °C in a water bath with gentle shaking. Following incubation, tubes were dipped in a cryogenized ice bath and placed in a freezer. Then, the frozen medium containing the pollen grains was lyophilized and stored at –80 °C until further analysis. The data were expressed on the basis of pollen weights before prehydration.
Reverse transcription-polymerase chain reaction (RT-PCR)
Total RNAs in the pollen grains before and after incubation for 20 min was extracted with the TRIZOL reagent system (Invitrogen Corp., Carlsbad, CA, USA) according to the manufacturer's instructions. The extract was DNA-decontaminated with RNase-free DNase I (Invitrogen Corp.), diluted with RNase-free water and then used for relative quantitative RT-PCR to monitor ADC and SAMDC gene transcription. First-stranded cDNAs were synthesized from 3 µg of total RNA using the Superscript First-strand Synthesis System (Invitrogen Corp.) with random hexamers as primers. PCR was performed in a thermal cycler (model 480, TaKaRa Biomedical, Tokyo, Japan) using gene-specific primers for ADC (forward primer: 5'-TCC TGT TGG CGA TGA ACT G-3'; reverse primer: 5'-CAG AGT AAA TCT GAG TGG CCT CAC-3') and SAMDC (forward primer: 5'-TCT GCC ATT GGT TTT GAA G-3'; reverse primer: 5'-GGG TAT ACC TCA CGT CTT G-3'), respectively (Antognoni et al., 2002). The reaction mixture (50 µl) contained 2 µl of the first-stranded reaction, 1 µmol TRIS–HCl (pH 8.0), 125 nmol KCl, 10 nmol each of dATP, dCTP, dGTP, and dTTP, 2.5 U Taq DNA polymerase (Invitrogen Corp.), 40 pmol each of gene specific primers, and 20 pmol of PCR internal standards. The PCR standard used was QuantumRNA Universal 18S Internal Standards (Ambion, Inc., Austin, TX, USA) with the molar ratio of 18S primer pairs to competimers at 1:9 for ADC and QuantumRNA Classic 18S internal standards (Ambion) with the molar ratio of 18S primer pairs to competimers at 3:7 for SAMDC.
The PCR reaction was performed with an initial denaturing step at 95 °C for 5 min, followed by 25 cycles of 95 °C for 1 min, 54 °C (SAMDC) or 55 °C (ADC) for 1 min, and 72 °C for 2 min, and a final termination step of 10 min at 72 °C. After amplification, the PCR products were resolved by electrophoresis on a 3% (w/v) agarose gel and stained with 0.1% (w/v) ethidium bromide. The stained gel was digitally photographed using a digital imaging system (model FAS-III; Toyobo, Osaka, Japan). Scion Image Windows program (Scion, http://www.sciosorp.com) was used to quantify the band intensity.
Immunoblot analysis
Soluble proteins in the pollen grains before and after incubation for 20 min were extracted with TRIS–HCl (pH 8.0) containing 1% (v/v) 2-mercaptoethanol, one tablet per 10 ml of ‘complete Mill’ protease inhibitor (Roche, Mannheim, Germany) with 1 mM EDTA. The protein concentration in the extract was determined using a protein assay reagent (Bio-Rad, Hercules, CA, USA). For immunoblot analysis, 30 µg or 40 µg of proteins for each lane was electrophoresed by SDS–PAGE and then blotted onto PVDF (polyvinylidene difluoride) membranes (ATTO, Tokyo, Japan). Protein was transferred to PVDF membranes and reacted with a polyclonal antibody for ADC and SAMDC. Blots were developed using horseradish peroxidase coupled to goat anti-rabbit IgG and goat anti-guinea pig IgG (ICN, Aurora, OH, USA) for ADC and SAMDC detection, respectively, with 3,3-diaminobenzidine tetrahydrochloride (ICN) as a colorgenic substrate.
The ADC-specific antibody was raised in a rabbit against a synthetic oligopeptide corresponding to the N-terminus of ADC (CESSLPLHEIGSGDGGRYY) (TaKaRa Biomedical). To prepare the antibody for SAMDC, proteins were extracted from 250 g of tomato flowers and SAMDC was purified by affinity chromatography as described by Hiatt et al. (1986). SAMDC-specific antibody was raised in a guinea pig against the purified SAMDC proteins (TaKaRa Biomedical).
Assay of protein synthesis
Pollen grains (8 mg) were prehydrated as above and incubated in 5 ml of germination medium containing 29.5 kBq ml–1 of [14C]amino acid mixture ([U-14C]protein hydrolysate; Amersham Pharmacia Biotech, Piscataway, NJ, USA) and 10 µg ml–1 of carrier casein hydrolysate (ICN), which was supplemented or not with 1 mM Spd. Following incubation for 60 min, 5 ml of 10% trichloroacetic acid was added to the medium to terminate protein synthesis. Then, to facilitate protein coagulation, the medium was mixed with 100 µl of 0.3% (w/v) bovine serum albumin and left at room temperature for 30 min. Following centrifugation at 14 000 g for 10 min, the pellet was repeatedly washed with 5% trichloroacetic acid to remove radiolabelled non-proteinous compounds. Then, the pellet was taken to dryness with a stream of N2 and homogenized with 1 ml of 100 mM TRIS–HCl (pH 8.0) containing 0.1 mM sodium dodecyl sulphate and 1 mM phenylmethylsulphonyl fluoride using a hand homogenizer. After centrifugation at 20 000 g for 20 min, 0.5 ml of the supernatant was taken in a vial, mixed with 4 ml of scintillation cocktail (Scintisol 500; Wako Pure Chemical, Tokyo, Japan), and the radioactivity was counted with a liquid scintillation counter.
Statistical analysis
All assays were repeated at least three times, excepting the RT-PCR and immunoblot analyses that were repeated only once. The data were statistically analysed by Tukey's test using SPSS/PC version 12.0 (SPSS Inc., Chicago, IL, USA).
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Results |
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The germinability of pollen grains collected in the spring or autumn of the years 2000–2004 was evaluated at the time of mixing the daily collected pollen grains (about 10 d after the beginning of daily pollen collection). Their germination percentages were around 65%, irrespective of the season of pollen collection, indicating the similarity of the potential activity of pollen grains collected in different seasons.
The germination percentage of 1-month-old pollen grains measured at 10 d after the beginning of pollen collection was 67% (Fig. 1). This germinability declined progressively as the storage duration exceeded 24 months; after 42 months three-quarters of originally viable pollen grains lost germinability and even the viable pollen grains showed a reduced capacity for tube elongation. The addition of 1 mM Put, Spd, or Spm to the germination medium markedly promoted germination and particularly tube elongation exclusively in pollen grains stored for longer than 24 months. The fertilization ability of the pollen also decreased in inverse proportion to the storage duration (Fig. 2). Here also, impregnation of pollen grains with Spd restored the fertilization ability of the aged pollen. The fresh weight (200–210 g) and seed number (150–180) of fruit did not differ with pollen age and Spd treatment.
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These results provoked the hypothesis that the aged pollen might be deficient in endogenous polyamines necessary for germination and tube growth to proceed normally. Thus, polyamine contents and polyamine biosynthetic enzyme activities were analysed in 1-, 24-, and 42-month-old pollen grains before and after incubation for 60 min. The results showed that, before incubation, pollen grains of different ages had similar contents of Put, Spd, and Spm in all of the free, conjugated, and bound forms (Fig. 3). After 60 min of incubation, 1-month-old pollen grains displayed a significant increase in free Put, Spd, and Spm, and bound Spd and Spm. However, such increases were much smaller in 24-month-old pollen grains and none of these polyamines increased during incubation in 42-month-old pollen grains. Enzyme analysis revealed that ADC and SAMDC activities of pollen before incubation did not differ with pollen age (Fig. 4). Following incubation, ADC and SAMDC activities increased 4.0- and 6.9-fold, respectively, in 1-month-old pollen grains. In 24- and 42-month-old pollen grains, however, the rates of increase in these enzyme activities were only 1.7- and 1.1-fold for ADC and 2.1- and 1.7-fold for SAMDC, respectively, indicating the age-dependent deprivation of pollen capacity to enhance polyamine biosynthetic enzyme activities upon rehydration.
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Changes in the levels of ADC and SAMDC mRNAs and their translation products in the pollen during germination were examined by RT-PCR and immunoblot analyses, respectively. The incubation period was set at 20 min in order to observe the early events of ADC and SAMDC gene expression in tomato pollen. Before rehydration, 1-, 24-, and 42-month-old pollen grains showed similar levels of gene transcripts for both enzymes and the levels did not change during the 20 min incubation period, irrespective of pollen age (Fig. 5). In the immunoblot analysis, both ADC and SAMDC antibodies cross-reacted with two polypeptides (Fig. 6); 68 kDa and 40 kDa bands correspond to proenzymes and 26 kDa and 28 kDa bands to active enzymes for ADC and SAMDC, respectively (Kashiwagi et al., 1990; Malmberg and Cellino, 1994). The content of active enzyme proteins in the pollen before incubation did not differ with the duration of pollen storage in both ADC and SAMDC. At the end of the 20 min incubation, 1-month-old pollen grains displayed 2- and 1.7-fold increases in ADC and SAMDC protein levels, respectively. However, the increase in these enzyme protein levels was considerably diminished in 24-month-old pollen grains and did not occur in 42-month-old pollen grains. Levels of proenzymes showed a similar trend to those of active enzymes.
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Polyamines are known to affect in vivo as well as in vitro protein synthesis in plants (Childs et al., 2003; Bachrach, 2005). On the other hand, bicellular pollen undergoes a rapid increase in the rate of protein synthesis during the early phase of germination (Mascarenhas and Bell, 1969; Hoekstra and Bruinsma, 1979). Hence, the protein-synthesizing capacity of 1-, 24-, and 42-month-old pollen grains was estimated in the presence or the absence of 1 mM Spd in the germination medium. In the absence of added Spd, the amount of proteins synthesized in the pollen during the 60 min incubation period was reduced as the storage duration was increased over 24 months (Fig. 7). It is interesting to note that the addition of Spd to the incubation medium enhanced protein synthesis exclusively in the aged pollen grains, and, in consequence, the difference in the rate of protein synthesis between the pollen of different storage durations became very small. As a whole, the rate of protein synthesis in the pollen was exponentially correlated with germination percentage (R2=0.795) and tube length (R2=0.924). The addition to the germination medium of the protein synthesis inhibitor cycloheximide at 10 ng ml–1 inhibited germination and tube growth of 1-month-old pollen grains in the presence of 1 mM Spd in the medium and cancelled the germinability-restoring effect of Spd in 24- and 42-month-old pollen grains (Fig. 8).
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Discussion |
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In the present study, any attributes of non-stored fresh tomato pollen were unexamined. This is because a single tomato flower at anthesis has about 0.2 mg of dry pollen grains, and therefore it is usually necessary to store daily collected pollen grains in a freezer for at least several days until a sufficient quantity of pollen grains have been collected to carry out an assay. Generally, such a procedure may not affect viability and metabolic activity of the pollen (Hoekstra and Bruinsma, 1979; Hanna and Towill, 1995). In the present study, germination percentage of 1-month-old pollen grains was about 65%. This germination percentage is almost maximal for the fresh pollen grains of tomato cv. Jifan No. 3 at 25 °C in the present incubation system (Song et al., 2001, 2002). In addition, in the present study, exogenous polyamines were ineffective in promoting germination and protein synthesis of 1-month-old pollen grains. Moreover, there was a close correlation between germinability and the capacity of the pollen to synthesize protein. From these facts, it is inferred that the capacities of fresh tomato pollen to germinate and synthesize polyamines, polyamine biosynthetic enzymes, and proteins in general may be virtually the same as those of the 1-month-old pollen that was observed in the present study.
Tomato pollen showed a gradual decrease in germinability and fertilization ability when the storage duration was 24 months or longer (Figs 1, 2). This loss of pollen viability was accompanied by attenuation of the capacity to increase ADC and SAMDC activities and endogenous polyamine contents after rehydration (Figs 3, 4). It is generally accepted that, for pollen germination to take place normally, a considerable amount of polyamines has to be newly synthesized after rehydration, which requires enhanced activity of polyamine biosynthetic enzymes (Chibi et al., 1994; Song et al., 2001, 2002). Thus, it is inferred that the loss of viability of tomato pollen during storage involves reduction in the capacity to enhance polyamine biosynthetic enzyme activities upon rehydration. This view is supported by the fact that exogenous Put, Spd, and Spm applied to germination medium and Spd impregnated into pollen grains prior to pollination were effective in restoring germinability and fertilization ability, respectively, exclusively in aged pollen grains (Figs 1, 2). Nevertheless, restoration of their germinability and fertilization ability by exogenous polyamines was imperfect. This may suggest that attenuation of polyamine biosynthesis is not the sole cause for the loss of viability in aged tomato pollen.
In a previous study, exogenous Put failed to mitigate the inhibition of pollen germination induced by a SAMDC inhibitor while Spd and Spm reversed it, indicating that Put does not play an important role in germination of tomato pollen (Song et al., 2001). In the present study, however, Put did promote germination of aged tomato pollen similarly to Spd and Spm (Fig. 1). In view of the fact that even the aged pollen grains exhibited positive SAMDC activity (Fig. 4), it is possible that Put taken up by them was enzymatically converted to Spd and/or Spm, even at a reduced rate, to fulfil their requirement for pollen germination.
It has been shown that almost all mRNAs coding for proteins necessary for pollen germination and tube growth are already present in mature pollen grains and remain translationally inactive until they are rehydrated (Mascarenhas, 1993; Franklin-Tong, 1999). Accordingly, results of the RT-PCR analysis in the present study (Fig. 5) may indicate that ADC and SAMDC mRNAs present in mature tomato pollen are fully transcribed before and during pollen maturation, and hence the transcription process may not be involved in the limited increase of these enzyme activities upon rehydration in aged pollen grains compared with fresh ones. On the other hand, ADC and SAMDC mRNA translation was enhanced within 20 min after rehydration in 1-month-old tomato pollen. As the pollen-storage duration exceeded 24 months, however, translation of these mRNAs became sluggish in a similar manner to enzyme activity after incubation (Fig. 6). The difference in the degree of increases in enzyme protein levels (Fig. 6) and enzyme activities (Fig. 4) during incubation could simply be ascribable to the difference in the period of incubation (20 min versus 60 min). Thus, it is inferred that the impaired translation of ADC and SAMDC mRNAs is a major cause for the decreased capacity of aged tomato pollen to enhance these enzyme activities after rehydration. Despite a lot of evidence demonstrating that polyamine biosynthetic enzyme activities in plants are attenuated during ageing (Galston and Sawhney, 1990; Paschalidis and Roubelakis-Angelakis, 2005), this study is the first, as far as is known, to provide direct evidence that translation of polyamine biosynthetic enzyme mRNAs in plants is impaired with ageing. ADC and SAMDC mRNAs are characterized by possessing small upstream open reading frames in the relatively long 5' untranslated leader sequences. These small upstream open reading frames are considered to be responsible for translational up- and down-regulation of the downstream open reading frame coding for the enzyme (Chang et al., 2000; Hanfrey et al., 2003; Hu et al., 2005, Kusano et al., 2007). However, the mechanism by which tomato pollen has lost the capacity to translate polyamine biosynthetic enzyme mRNAs during storage remains to be investigated.
It was also indicated that the capacity of tomato pollen to synthesize proteins in general, rather than the capacity to translate specific mRNAs like ADC and SAMDC mRNAs, was progressively attenuated during the long-term dry storage (Fig. 7). Spd applied to the germination medium promoted protein synthesis and concurrently restored germinability solely of aged pollen. This effect of exogenous Spd in promoting germination of aged pollen grains was negated by cycloheximide (Fig. 8), indicating that one of the important roles of exogenous Spd in restoring germinability of aged tomato pollen is to stimulate protein synthesis. It is well documented that the active synthesis of many novel proteins in rehydrated pollen grains is an important factor for the occurrence of normal pollen germination in many bicellular pollen species (Hoekstra and Bruinsma, 1979; Mascarenhas, 1993). Therefore, it is concluded that the decline in overall protein synthesis is a major cause of viability loss of pollen during long-term dry storage and inadequate activation of polyamine biosynthetic enzymes resulting in deficiency of polyamines (Spd and Spm) is primarily responsible for the decline in protein synthesis in aged tomato pollen.
It has been shown that ribosomes in bicellular pollen have to undergo a developmental change from monosomes to polysomes, following rehydration, prior to commencing active protein synthesis (Cresti et al., 1977; Tupy, 1977; Hoekstra and Bruinsma, 1979). There are many reports demonstrating important roles of polyamines (Spd and Spm in particular) in RNA synthesis and polysome assembly (Kakegawa et al., 1986; Huang et al., 1990; Bachrach, 2005). Thus, it seems likely that the increased Spd of exogenous origin in aged tomato pollen cells serves to organize polysomes, a prerequisite for active protein synthesis in the pollen and eventually pollen tube emergence. Another role of increased Spd could be associated with protein synthesis per se. It is widely recognized that initiation of protein synthesis in eukaryotic cytosol is facilitated by a number of auxiliary protein factors referred to as eukaryotic translation initiation factors (eIFs) (Melefors and Hentze, 1993; Dever, 2002). One such protein is eIF5A (formerly eIF4D), which has been implicated in not only translation initiation but also mRNA metabolism and ribosome biogenesis (Valentini et al., 2002; Childs et al., 2003). This protein is post-translationally modified by the addition of a butylamine residue to a highly conserved lysine of inactive eIF5A, giving rise to the formation of an unusual amino acid, hypusine, and activated eIF5A (Park, 2006). The butylamine residue is exclusively derived from Spd and therefore hypusine synthesis is impaired in Spd-deficient cells (Gerner et al., 1986; Chattopadhyay et al., 2003). An arrest in cell proliferation induced by a SAMDC inhibitor has been attributed to depletion of eIF5A following deprivation of Spd (Byers et al., 1994). Besides, Spd can stimulate aminoacyl-tRNA synthetase activity and thereby promote peptide chain elongation (Mukhopadhyay and Ghosh, 1986; Giannakouros et al., 1990). Also, Spm is known to stimulate protein synthesis in biological and cell-free systems (Marcu and Dudock, 1974; Gross and Rubino, 1989; Bachrach, 2005). Thus, it seems plausible that exogenously augmented Spd and Spm restored germinability of aged tomato pollen through affecting these polyamine-related affairs in RNA and protein synthesis. A portion of absorbed Spm could have played its role after being converted to Spd through the acetylation mechanism (Bagni and Tassoni, 2001), as evidenced in animal cells (Byers et al., 1994). As stated above, Put per se is considered to be irrelevant to germination of tomato pollen (Song et al., 2001). In this respect, whether this trait of Put is ascribable for the inability of aged tomato pollen to promote protein synthesis requires further study.
Tomato pollen usually commences tube emergence at around 20 min after rehydration (Song et al., 2001). Therefore, results of the ADC and SAMDC mRNA translation experiment (Figs 5, 6) suggest that polyamine biosynthesis in tomato pollen is activated in advance of germination in order to make good use of polyamines for the synthesis of various kinds of proteins necessary for pollen germination. Here, a question arises as to whether the increased protein synthesis in aged tomato pollen by exogenous Spd is accompanied by the active synthesis of proteins that are not synthesized or synthesized only slightly in the absence of exogenous Spd. Clarification of this issue may help better understanding of the role of Spd in pollen germination processes.
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Footnotes |
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* Present address: Department of Life Science, College of BioScience & Bioengineering, Hebei University of Science & Technology, Shijiazhuang, Hebei 050018, China.
Present address: Laboratory of Plant Biotechnology, Faculty of Agriculture, Tokyo University of Agriculture, Atsugi, Kanagawa 243-0034, Japan.
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Abbreviations |
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ADC, arginine decarboxylase; eIF, eukaryotic translation initiation factor; Put, putrescine; RT-PCR, reverse transcription-polymerase chain reaction; SAMDC, S-adenosylmethionine decarboxylase; Spd, spermidine; Spm, spermine.
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References |
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