Journal of Experimental Botany, Vol. 52, No. 354, pp. 67-75,
January 2001
© 2001 Oxford University Press
Original Papers |
A ß-galactosidase-like gene is expressed during tobacco pollen development
School of Biosciences, Cardiff University, PO Box 915, Cardiff CF10 3TL, UK
Received 18 January 2000; Accepted 18 August 2000
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Abstract |
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cDNA clone (TP5) with significant homology to ß-galactosidases has been isolated from a mature tobacco pollen cDNA library by differential screening. The predicted protein of 715 aa shows high levels of homology to plant ß-galactosidases expressed during fruit ripening and senescence. Northern analysis shows that the TP5 transcript is expressed exclusively in developing anthers and mature pollen. The transcript is present at very low levels at meiosis and increases dramatically, late in microspore development after mitosis suggesting that the primary role for the protein is during pollen tube growth. ß-galactosidase activity, measured by scanning densitometry of histochemically stained tobacco microspores, is first detectable in the early to mid-vacuolate stage, and reaches a peak at microspore mitosis, thereafter decreasing as the microspores reach maturity. Southern analysis indicates that the TP5 gene is present in two copies, probably corresponding to the two ancestral genomes of N. tabacum.
Key words: Pollen, microsporogenesis, ß-galactosidase, gene expression.
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Introduction |
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The male gametophyte of flowering plants is a highly specialized tissue with essential functions in the recognition of compatible stigma and delivery of sperm cells to the ovule (Cheung, 1996
). To perform these functions, a unique pattern of gene expression is required comprising both messages specific to this stage of development and those shared with other tissues (Taylor and Hepler, 1997). The mature pollen grain stores messages in readiness for pollen germination, thus the majority of messages isolated from mature pollen will encode proteins needed during pollen tube growth (Mascarenhas, 1993). Pollen tubes extend by tip growth and require a rapid turnover of the tip wall materials; this is achieved by the delivery of pectin containing vesicles (dictyosomes) to the growing tip (Steer and Steer, 1989). Genes expressed during pollen development have been classed as ‘early’ and ‘late’ genes (Mascarenhas, 1990). Transcripts of early genes are first expressed just after meiosis, declining before pollen maturity, whereas late genes are expressed after microspore mitosis reaching a maximum in mature microspores. In some cases expression in the pollen tube has also been demonstrated (Kononowicz et al., 1992). Several of the late genes encode proteins putatively involved in pectin metabolizm, and include to date, polygalacturonase (Brown and Crouch, 1990; Niogret et al., 1991; Rogers and Lonsdale, 1992), pectate lyase (Wing et al., 1989; Rogers et al., 1992) and pectin methyl esterase (Mu et al., 1994; Wakeley et al., 1998). Pectin methyl esterases catalyse the de-methylation of pectin providing a suitable substrate for the action of polygalacturonase and pectate lyase (Lee and Macmillan, 1970). In addition it has been suggested that pectin methyl esterase may play a critical role in pollen tube growth by mediating the action of Ca2+on tube elongation (Nari et al., 1991). Enzyme activities for both polygalacturonase and pectin methyl esterases have been detected in pollen and or inflorescence tissue (Pressey and Reger, 1989; Richard et al., 1994), whereas the presence of pectate lyase activity has not been confirmed. ß-galactosidase activity has also been detected in Brassica pollen (Singh and Knox, 1985; Singh et al., 1985), but to date no sequence information for genes encoding this protein in pollen were available. ß-galactosidases act on various substrates including arabinogalactans, galactolipids and pectin to release galactose (Dey and Del Campillo, 1984) and are thought to play a role in cell wall loosening (Dopico et al., 1989). A gene expressed late in pollen development with strong homology to ß-galactosidases found in other plant tissues is described here: this is the first reported sequence likely to encode this enzymatic activity in pollen.
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Materials and methods |
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Isolation of cDNA clone and sequencing
A Nicotiana tabacum var. Samsung mature pollen cDNA library was constructed in
gt10 (Rogers and Lonsdale, 1992) and screened using mature pollen and leaf cDNA probes (Rogers et al., 1992). Clones hybridizing to the pollen, but not the leaf probe were selected for further analysis.
cDNA clone TP5 (2159 bp) was sequenced in both directions using an ABI 377 automated sequencer by creating a number of restriction enzyme sub-clones, ExoIII deletions (Henikoff, 1987), and designing specific primers. Two rounds of 5' RACE (Frohman et al., 1988) were used to obtain the 5' end of the cDNA, which was also sequenced in both directions.
Northern and Southern analysis
Formaldehyde gels for Northern analysis were as described previously (Rogers et al., 1992) using 15 µg of total RNA isolated using Tri-Reagent (Sigma). Equal loading was checked by ethidium bromide staining of agarose gels. For Southern analysis, high molecular weight DNA was extracted according to the protocol described previously (Doyle and Doyle, 1987), digested with restriction enzymes and electrophoresed through 0.8% agarose gels. RNA and DNA were transferred to nylon membranes by capillary blotting. Prehybridization and hybridization were performed at 60 °C in a solution containing 5xDenharts, 6xSSC, 0.1% SDS, 5% PEG, 0.1% tetrasodium pyrophosphate, and 100 µg ml-1 denatured herring sperm DNA. Random primed probes were prepared as described previously (Feinberg and Vogelstein, 1983). The probe used for the Northern and Southern blots was the 2159 bp cDNA clone which contains most of the ORF and the 3'UTR. Washes were in 2xSSC at 60 °C.
Enzyme cytochemical analysis of ß-galactosidase activity
Cytochemical assays for ß-galactosidase activity in developing microspores, mature and germinated pollen, were carried out essentially as previously described (Singh et al., 1985). Anthers were teased open to release the developing microspores, and transferred to a solution containing 50 mM sodium acetate pH 4.8, 2 mM potassium ferrocyanide and potassium ferricyanide, 5% (w/v) sucrose, amd 3 mM 5-bromo-4-chloro-3-indoxyl-ß-D-galactoside (in 50 µl N,N-dimethyl-formamide mg- of substrate). After incubation at 37 °C for 15 min, microspores were pelleted by centrifugation washed in PBS and the intensity of staining was measured using a Vickers M85A Integrating Microdensitometer at a wavelength of 550 nm. 15 microspores were assayed for each stage.
Developmental stages of the microspores were checked by fixing anthers in 3:1 absolute ethanol:galcial acetic acid for 2 h, followed by washes in 70% ethanol and water, and stained with 4,6-diamidino-2-phenylindole (DAPI) (0.5 µg ml-1) (Singh et al., 1985).
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Results |
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Isolation, structure and deduced amino acid sequence of the pollen specific cDNA clone TP5
Twenty clones were isolated by differential screening of a tobacco mature pollen cDNA library, using pollen and leaf probes. The clones fell into ten homology groups (Rogers and Lonsdale, 1992
), and the longest clone from each group was end sequenced. Sequences were compared to current databases and one clone with an insert size of 2.1 kb showed significant homology to ß-galactosidases. This cDNA was fully sequenced in both directions (2159 bp) and comparison to published ß-galactosidase sequences, suggested that the 5' portion was missing. Two rounds of 5' RACE were used to obtain the missing 5' end of the cDNA (598 bp) and the clones were fully sequenced. The overlapping regions between the three clones were 100% homologous confirming that the portions derived from the same mRNA. (The nucleotide sequence data reported here will appear in the EMBL, Genbank and DDBJ Nucleotide Sequence Databases under Accession number AJ250431.)
Analysis of the sequence revealed a single open reading frame of 2148 bp, 107 bp of 5' and 468 bp of 3' non-coding sequence. The putative protein is of 715 amino acids, has a predicted molecular mass of 81.32 kDa and a predicted pI of 8.5. The N terminal region is relatively hydrophobic and a transit peptide of 20 amino acids is predicted (von Heijne, 1983). This is similar in length to transit peptides of other pollen specific proteins which are thought to be exported (Rogers et al., 1992; Tebbutt et al., 1994) The context of the putative ATG start codon fits well with consensus sequences (Lutcke et al., 1987) and a polyadenylation site motif conforming to the consensus AATAAA is present 21 bp upstream from the poly(A) addition site. This short distance is similar to the 16 bp found in the maize pollen specific gene ZmC5 (Wakeley et al., 1998), but differs from other pollen specific transcripts which have longer than average distances between the AATAAA motif and poly(A) addition site (Tebbutt et al., 1994).
TP5 shows similarity to ß-galactosidases
The predicted amino acid sequence of TP5 revealed a high level of homology to plant (between 35.8% and 43.6%) and mammalian (19.8–20.2%) ß-galactosidases, but relatively less homology to fungal (10.3–16.3%) and bacterial ß-galactosidases (e.g. only 10.2% homology to E. coli ß-galactosidase). A phylogenetic tree was constructed using the Neighbour–Joining method (Fig. 1) and shows clearly that TP5 is most homologous to the plant ß-galactosidases, but forms a distinct sub-group. Six of the seven homology domains identified between the Xanthomonas gene and eukaryotic ß-galactosidases (Taron et al., 1995) are present in TP5 (Fig. 2) including all but one of the invariant amino acid positions identified previously (Taron et al., 1995). Two glutamic acid residues have been identified in the Xanthomonas gene as the potential active site nucleophile from homology to plant ß-1-3 glucanases for which the crystal structure is available (Varghese et al., 1994). These are also present in TP5 at amino acid positions 182 and 184. The putative proton donor site in TP5 is at amino acid 255. The highest level of homology between TP5 and other plant ß-galactosidase-like sequences is towards the N-terminal end of the predicted protein including the putative active site region (Domains 1 and 2).
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TP5 transcript is expressed exclusively in anthers and increases dramatically after microspore mitosis
Northern analysis using 15 µg of total RNA from nine tobacco tissues confirmed the tissue specificity of TP5. A transcript of approximately 3 kb was detected only in mature pollen (Fig. 3A), with no detectable expression in the other tissues analysed. Expression during anther development was also determined by Northern analysis (Fig. 3B). Total RNA was extracted from anthers taken from flowers at five stages of development based on bud length (Kyo and Harada, 1986; Koltunow et al., 1991), corresponding to Stage 1: 5–8 mm (meiosis/tetrads), Stage 2: 10–12 mm (pre-mitosis), Stage 3: 12–15 mm (microspore mitosis), Stage 4: 17–22 mm (early–mid-binucleate), Stage 5: 25–45 mm (mid–late-binucleate). The developmental stage was verified by DAPI staining (data not shown). A very weak signal was detectable from the earliest stage of anthers analysed, but a dramatic increase is evident between early–mid-binucleate to mid–late-binucleate, suggesting the majority of the expression is occurring well after microspore mitosis.
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Gene copy number of TP5
The gene family structure was investigated using the TP5 clone as a probe on a Southern blot of tobacco genomic DNA digested with EcoRI, EcoRV and HindIII (Fig. 4). There is one EcoRV site within the cDNA portion used as a probe and no sites for EcoRI or HindIII, although it is not known if there are any introns in this gene. At medium stringency conditions (2xSSC, 60 °C) two major fragments and two or three minor fragments hybridize. This suggests that there are one or two copies of the TP5 gene in the tobacco genome and two to three further copies of more distantly related sequences.
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Re-screening of the cDNA library, using clone TP5 identified five further homologous clones. Alignment of the 3' untranslated region revealed two groups sharing 92% homology. These are likely to correspond to the two ancestral genomes of N. tabacum: N. sylvestris and N. tomentosiformis and supplies further evidence for a copy number of two.
ß-galactosidase activity is detectable in developing pollen grains from just before microspore mitosis
Developing microspores were taken from anthers at five developmental stages as described above for the Northern analysis. Pollen from mature anthers was germinated as described earlier (Tebbutt et al., 1994). Microspores were stained using DAPI to determine the developmental stage, and used for comparative cytochemical analysis of ß-galactosidase activity essentially as described previously (Singh et al., 1985). (Fig. 5A). ß-galactosidase activity is first detectable just before microspore mitosis, and reaches a peak at microspore mitosis, thereafter decreasing as the microspores reach maturity. ß-galactosidase activity is also clearly present in the pollen tube cytoplasm throughout the length of the tube (Fig. 5B), however, it was not possible to compare the level of activity to the microspores by the method used. Little or no staining was apparent in the pollen or tube walls.
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Discussion |
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ß-Galactosidase catalyses the cleavage of ß-galactosidic linkages releasing galactose. ß-galactosidase activity has been detected in a wide range of plant species and tissues (Dey and Del Campillo, 1984
) and is thought to be involved in pectin, arabinogalactan and galactolipid metabolism. In fruit ripening, ß-galactosidases have been implicated in the loss of D-galactose residues from the cell wall. Genes encoding putative and confirmed plant ß-galactosidases have been isolated from ripening fruit: tomato (Carey et al., 1995) and apple (Ross et al., 1994), and senescent tissue: asparagus spears (King and Davies, 1995) and dianthus petals (Raghothama et al., 1991). The levels of homology between the TP5 cDNA and these plant genes, strongly suggests that TP5 may encode a ß-galactosidase enzyme. This is further supported by a comparison of amino acids thought to be important in the active site of ß-galactosidases. These comprise three glutamic acid residues identified in the Xanthomonas ß-galactosidase gene as the potential active site nucleophiles and proton donor (Varghese et al., 1994). Other characteristics of the predicted TP5 protein also confirm the close similarity to other ß-galactosidase genes. The predicted protein size of TP5, 715 aa, is similar to the plant ß-galactosidase-like genes reported to date, though slightly smaller (other plant ß-galactosidase-like genes range from 731 to 835 aa), however mammalian ß-galactosidases are smaller still. The leader sequence of TP5 is within the range (15–23 bp) of the other plant ß-galactosidase-like genes reported and suggests that this protein might also be exported. ß-galactosidase activity has been previously determined in Brassica pollen (Singh et al., 1985) and shown to be located both cytoplasmically and extracellularly, implying export of this protein. It has also been reported in the cytoplasm and walls of pollen and pollen tubes of Pyrus and the cytoplasm of Amaryllis (Villar et al., 1993). In tobacco, the histochemical data suggest that the enzyme is mainly located in the cytoplasm and inner wall or plasma membrane of the pollen grain and throughout the cytoplasm of the tube with little or no staining in the wall (Fig. 5B
). The histochemical data are therefore at variance with the sequence data supporting an exported protein. Although some blue coloration of the pollen germination medium was detected (data not shown), this could be due to damaged tubes or grains in the preparation. Further biochemical assays might be helpful to resolve this point, and further quantify the expression.
ß-Galactosidase activity was detectable in developing tobacco microspores in early to mid vacuolate microspores and reached a peak around microspore mitosis, thereafter falling off as the microspores reached maturity. This profile is very similar to that determined for Brassica campestris (Singh et al., 1985), although in Brassica, which has tricellular pollen, the peak was reached late in generative cell mitosis. A comparison of TP5 expression at the transcriptional level with the activity of ß-galactosidase in developing microspores reveals a discrepancy in the timing of these events. TP5 transcript is detectable by Northern analysis at only very low levels until well after pollen mitosis. This pattern of accumulation is very similar to that found in other ‘late’ pollen specific genes such as the pectate lyase-like genes in both tobacco and tomato (Wing et al., 1989; Rogers and Lonsdale, 1992). Singh et al. suggest that the fall in ß-galactosidase activity late in Brassica microspore development might be related to compartmentalization of the enzyme in preparation for the period of dormancy in mature pollen grains, making accurate determination of the activity difficult (Singh et al., 1985).
Substantial levels of ß-galactosidase activity in pollen tubes were clearly detected, suggesting that the enzyme activity may be required during tube growth. Earlier enzyme activity may have a different function during microspore development. A dual role is also likely for other microspore-expressed genes involved in pectin metabolism. For example, two genes with homology to pectin methylesterases isolated in petunia and maize are transcribed late in pollen development (Mu et al., 1994; Wakeley et al., 1998). Another homologous gene, however, identified in Brassica (Albani et al., 1991), is classed as an ‘early’ gene and is expressed from tetrads through mitosis I to bi-nucleate pollen, but is not detectable in mature tri-nucleate pollen. Also a low expression level of the putative pectate lyase gene in tobacco (Rogers et al., 1992), is detectable by Northern and promoter expression analysis early in pollen development as well as a much higher expression at later stages (HJ Rogers et al., unpublished results). This suggests the possibility that some late pollen genes might be active early in microspore development. The failure to detect TP5 transcripts at early stages of microsporogenesis might reflect a lack of sensitivity of the Northern analysis. However, it is also possible that the ß-galactosidase activity measured early in microspore development does not reflect expression of TP5, but derives from expression of other related ß-galactosidase genes in tobacco.
Southern data on TP5 strongly suggests that this gene is present in two copies in the N. tabacum genome probably derived from the two ancestral species. Evidence from mutant analysis in oil seed rape (Singh and Knox, 1985) indicates that in this species pollen ß-galactosidase activity is encoded by a single gene. This supports the findings of this study in tobacco. However, ß-galactosidase activity is also found in other plant tissues such as ripening fruit and senescent tissues thus other genes encoding ß-galactosidase enzymes must be present in the tobacco genome. It is possible that the weaker signals detected on our Southern blot correspond to these other genes.
The putative ß-galactosidase described in this paper is an additional member of a set of genes whose mRNA accumulates late in pollen development, and is presumed to be stored in readiness for pollen germination. Some of these genes are thought to be involved in pectin metabolism including polygalacturonase, pectin methylesterase and pectate lyase. These latter enzymes probably play an important role in pollen tube wall turnover, however, it is unclear from these histochemical data whether ß-galactosidase activity might also be involved in pollen wall metabolism since staining in the pollen and tube walls was not detected. Singh and Knox postulated a role for the Brassica pollen ß-galactosidase in the hydrolysis of arabinogalactan (Singh and Knox, 1985). Recent reports suggest that pollen tube-mediated de-glycosylation of arabinogalactan stylar proteins may be important in guiding pollen tubes in compatible pollination events (Cheung et al., 1995; Wu et al., 1995), thus if exported, pollen tube ß-galactosidase might also be involved in pollen tube guidance.
Mutants defective in ß-galactosidase isolated in Brassica campestris, have impaired fertility (Singh and Knox, 1985) suggesting an important role for this enzyme in pollen tube growth or fertilization. This contrasts with experiments to down-regulate some of the other putative pectin-metabolizing genes by antisense technology, which produced no detectable effects on fertility (McCormick et al., 1991; DM Lonsdale, personal communication).
Switching of ß-galactosidase activity has also been implicated in a change in the phase of Populus pollen tube growth in vivo (Villar et al., 1993). In this species there appear to be two physiological phases in the growth of pollen tubes in vivo. During the first stage the tube is considered to be essentially autotrophic, deriving its nutrients from stored reserves of starch laid down during the formation of the mature pollen grain. However, in compatible pollinations the physiology of the pollen tube shifts to a heterotrophic mode of nutrition requiring the synthesis of new proteins involved in uptake of nutrients from the transmissive tract of the stigma. Increased activity of ß-galactosidase is associated with this change in Populus, which may have important implications for the success of a pollination event and be the result of signalling between the style and the pollen-tube.
For the role of TP5 during pollen tube growth in tobacco to be characterized fully, it will be necessary to confirm that the TP5 gene product has ß-galactosidase activity by in vitro expression studies, establish the cellular or extracellular localization of the protein and identify its substrate specificity. This work is currently in progress.
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Acknowledgments |
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We wish to thank Dr DM Lonsdale (John Innes Centre, Norwich) in whose laboratory clone TP5 was originally isolated. We would also like to thank Dr S Hinsull and G Lewis for assistance with the sequencing, and Dr D Francis for assistance with the microdensitometry.
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Notes |
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1 To whom correspondence should be addressed. Fax: +44 2920 874305. E-mail: rogershj{at}cf.ac.uk
2 Present address: Institute of Medical Genetics, University of Wales, College of Medicine, Heath Park, Cardiff CF4 4XN, UK.
3 Present address: KS-Biomedix, 6 Romans Business Park, East Street, Farnham, Surrey GU9 7SX, UK.
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