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• W2077828827 abstract "The surface of a pollen grain consists of an outermost coat and an underlying wall. In maize (Zea mays L.), the pollen coat contains two major proteins derived from the adjacent tapetum cells in the anthers. One of the proteins is a 35-kDa endoxylanase (Wu, S. S. H., Suen, D. F., Chang, H. C., and Huang, A. H. C. (2002) J. Biol. Chem. 277, 49055–49064). The other protein of 70 kDa was purified to homogeneity and shown to be a β-glucanase. Its gene sequence and the developmental pattern of its mRNA differ from those of the known β-glucanases that hydrolyze the callose wall of the microspore tetrad. Mature pollen placed in a liquid medium released about nine major proteins. These proteins were partially sequenced and identified via GenBank™ data bases, and some had not been previously reported to be in pollen. They appear to have wall-loosening, structural, and enzymatic functions. A novel pollen wall-bound protein of 17 kDa has a unique pattern of cysteine distribution in its sequence (six tandem repeats of CX 3CX 10–15) that could chelate cations and form signal-receiving finger motifs. These pollen-released proteins were synthesized in the pollen interior, and their mRNA increased during pollen maturation and germination. They were localized mainly in the pollen tube wall. The pollen shell was isolated and found to contain no detectable proteins. We suggest that the pollen-coat β-glucanase and xylanase hydrolyze the stigma wall for pollen tube entry and that the pollen secrete proteins to loosen or become new wall constituents of the tube and to break the wall of the transmitting track for tube advance. The surface of a pollen grain consists of an outermost coat and an underlying wall. In maize (Zea mays L.), the pollen coat contains two major proteins derived from the adjacent tapetum cells in the anthers. One of the proteins is a 35-kDa endoxylanase (Wu, S. S. H., Suen, D. F., Chang, H. C., and Huang, A. H. C. (2002) J. Biol. Chem. 277, 49055–49064). The other protein of 70 kDa was purified to homogeneity and shown to be a β-glucanase. Its gene sequence and the developmental pattern of its mRNA differ from those of the known β-glucanases that hydrolyze the callose wall of the microspore tetrad. Mature pollen placed in a liquid medium released about nine major proteins. These proteins were partially sequenced and identified via GenBank™ data bases, and some had not been previously reported to be in pollen. They appear to have wall-loosening, structural, and enzymatic functions. A novel pollen wall-bound protein of 17 kDa has a unique pattern of cysteine distribution in its sequence (six tandem repeats of CX 3CX 10–15) that could chelate cations and form signal-receiving finger motifs. These pollen-released proteins were synthesized in the pollen interior, and their mRNA increased during pollen maturation and germination. They were localized mainly in the pollen tube wall. The pollen shell was isolated and found to contain no detectable proteins. We suggest that the pollen-coat β-glucanase and xylanase hydrolyze the stigma wall for pollen tube entry and that the pollen secrete proteins to loosen or become new wall constituents of the tube and to break the wall of the transmitting track for tube advance. In plants, sexual reproduction is initiated when the pollen grain (male component) lands on the stigma of the style (female component) in the flowers (for reviews, see Refs. 1Mascarenhas J.P. Plant Cell. 1993; 5: 1303-1314Crossref PubMed Scopus (363) Google Scholar, 2Taylor L.P. Hepler P.K. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1997; 48: 461-491Crossref PubMed Scopus (639) Google Scholar, 3Bewley J.D. Hempel F.D. McCormick S. Zambryski P. Buchanan B.B. Gruissem W. Jones R.L. Biochemistry and Molecular Biology of Plants. American Society for Plant Biologists, Rockville, MD2000: 988-1043Google Scholar). The pollen produces a tube that penetrates the stigma and grows through the wall of adjacent cells in the transmitting track in the style to reach the ovary, where the tube delivers the male gametes to fertilize the eggs. Pollen tube penetration of the stigma and advancement in the style are critical steps in sexual reproduction; yet biochemical information on this process is limited. The surface of a pollen grain makes the initial contact with the stigma. It includes an outermost coat and an underlying wall (1Mascarenhas J.P. Plant Cell. 1993; 5: 1303-1314Crossref PubMed Scopus (363) Google Scholar, 2Taylor L.P. Hepler P.K. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1997; 48: 461-491Crossref PubMed Scopus (639) Google Scholar, 3Bewley J.D. Hempel F.D. McCormick S. Zambryski P. Buchanan B.B. Gruissem W. Jones R.L. Biochemistry and Molecular Biology of Plants. American Society for Plant Biologists, Rockville, MD2000: 988-1043Google Scholar). The coat consists of lipids and proteins, which are initially synthesized and accumulated in the tapetum cells that enclose the pollen locule in the anthers. Upon lysis of the tapetum cells, the accumulated lipids and proteins are discharged onto the microspores (maturing pollen), forming the bulk of the pollen coat. In some species, the coat also contains minor proteins involved in self-incompatibility, which are synthesized in the pollen interior (4McCormick S. Curr. Opin. Plant Biol. 1998; 1: 18-25Crossref PubMed Scopus (23) Google Scholar). The underlying wall consists of sporopollenin and other polymers embedded with proteins; it is derived from the tapetum and the pollen interior (3Bewley J.D. Hempel F.D. McCormick S. Zambryski P. Buchanan B.B. Gruissem W. Jones R.L. Biochemistry and Molecular Biology of Plants. American Society for Plant Biologists, Rockville, MD2000: 988-1043Google Scholar). The pollen coat consists of lipids and proteins, and its composition varies, depending on the species. The coat in insect- or self-pollinating species, of which Brassica and Arabidopsis are the best studied (5Wu S.S.H. Platt K.A. Ratnayake C. Wang T.W. Ting J.T.L. Huang A.H.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12711-12716Crossref PubMed Scopus (96) Google Scholar, 6Piffanelli P. Ross J.H.E. nad Murphy D.J. Plant J. 1988; 11: 549-556Crossref Scopus (107) Google Scholar, 7Mayfield J.A. Fiebig A. Johnstone S.E. Preuss D. Science. 2001; 292: 2482-2485Crossref PubMed Scopus (178) Google Scholar), is thick; steryl esters and very non-polar lipids are the major lipids, and oleosins are the predominant proteins. The lipids are for waterproofing, whereas the amphipathic oleosins may act as a wick for water uptake to initiate germination. The pollen coat in wind-pollinating species is thin (3Bewley J.D. Hempel F.D. McCormick S. Zambryski P. Buchanan B.B. Gruissem W. Jones R.L. Biochemistry and Molecular Biology of Plants. American Society for Plant Biologists, Rockville, MD2000: 988-1043Google Scholar, 8Bih F.Y. Wu S.S.H. Ratnayake C. Walling L.L. Nothnagel E.A. Huang A.H.C. J. Biol. Chem. 1999; 274: 22884-22894Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), and major biochemical studies have been carried out only with maize (8Bih F.Y. Wu S.S.H. Ratnayake C. Walling L.L. Nothnagel E.A. Huang A.H.C. J. Biol. Chem. 1999; 274: 22884-22894Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 9Wu S.S.H. Suen D.F. Chang H.C. Huang A.H.C. J. Biol. Chem. 2002; 277: 49055-49064Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Maize pollen coat contains undefined neutral lipids and a few proteins of 25, 35, and 70 kDa. Only the 35-kDa protein, which is the most abundant, has been characterized and shown to be an active endoxylanase. It is initially synthesized as a large, inactive precursor in the tapetum and then activated after cleavage by a serine protease at both the N and C termini. The precursor or the mature 35-kDa xylanase does not have an apparent signal sequence that could target the protein to specific organelles; the protein is presumed to be present in the cytosol and is released to the anther locule and then the pollen surface after lysis of the tapetum cells. After landing on the stigma, the pollen would release the xylanase to hydrolyze the stigma wall for the entry of the pollen tube into the transmitting track. The characteristics of the other coat proteins of 70 and 25 kDa are presented in the current report. Imbibed pollen quickly releases proteins that were present in the coat or the wall, or are secreted from the pollen interior. These proteins include expansin, extensin, polygalacturonase, trypsin inhibitors, and a few others (2Taylor L.P. Hepler P.K. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1997; 48: 461-491Crossref PubMed Scopus (639) Google Scholar, 10Cosgrove D.J. Nature. 2000; 407: 321-326Crossref PubMed Scopus (1145) Google Scholar, 11Allen R.L. Lonsdale D.M. Plant J. 1993; 3: 261-271Crossref PubMed Scopus (77) Google Scholar, 12Cosgrove D.J. Bedinger P. Durachko D.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6559-6564Crossref PubMed Scopus (300) Google Scholar). Most of them are also categorized as pollen allergens because of their allergenic properties (13Andersson K. Lidholm J. Int. Arch. Allergy Immunol. 2003; 130: 87-107Crossref PubMed Scopus (302) Google Scholar). Some of these proteins were shown to have their mRNA present in mature pollen. The relative amounts of these individual proteins in the whole pollen are unknown. It is also unclear whether these proteins are present in the coat or the wall of the mature or germinated pollen and thus whether they will exert their functions on the stigma or inside the style. Another uncertainty is whether these proteins and their mRNA are present continuously in the pollen or pollen tube after germination. Finally, although these proteins have been individually studied in diverse species, their possible collaborative actions in one species has not been examined. All of these uncertainties are addressed in this report. We have used maize as an example of wind-pollinating species to characterize the major pollen proteins present in the coat and released from the wall or interior of mature and germinated pollen. The coat proteins include a xylanase and a β-glucanase derived from the adjacent tapetum cells. The pollen-released proteins represent about 10% of the total pollen proteins. They include many wall-reactive constituents and are synthesized in the pollen interior and secreted to the wall and the exterior. The actions of the coat and pollen-released proteins would allow the advance of the pollen tube on the stigma and through the style, respectively. Several of the proteins in the coat and released from the pollen are novel or have not been previously reported as being present in the pollen. Plant Materials—Maize (Zea mays L., B73) plants were grown in a greenhouse, and fresh pollen and anthers were collected as described (9Wu S.S.H. Suen D.F. Chang H.C. Huang A.H.C. J. Biol. Chem. 2002; 277: 49055-49064Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Anthers of five developmental stages were selected on the basis of the following criteria (8Bih F.Y. Wu S.S.H. Ratnayake C. Walling L.L. Nothnagel E.A. Huang A.H.C. J. Biol. Chem. 1999; 274: 22884-22894Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar): At stage 1, the tassel was still embedded in the shoot apex. The anthers filled up about one-third of each floret. Each microspore mother cell had undergone meiosis to produce a tetrad of microspores, which were still encased within a callose wall. At stage 2, the upper portion of the tassel had protruded from the shoot apex. The anthers filled up about one-half of the floret. Young microspores had been released from the dissolved callose wall, and the outer pollen wall (exine) had been synthesized. At stage 3, the tassel had protruded completely out of the top of the plant. The anthers filled up about two thirds of the floret. The microspores had become larger and contained multiple small vacuoles. The first mitosis had occurred, and the microspores were binucleate. At stage 4, the anthers filled up the floret completely. Second mitosis in the microspores had occurred, and the microspores were trinucleate. At stage 5, the tassel was yellow. Some of the florets on the tassel were open, and the pollen was ready to be released. Pollen Germination—A sample of 5 mg of freshly collected pollen was placed in 1.5 ml of liquid germination medium containing 100 ppm Ca(NO3)2, 10 ppm H3BO3, 37.5 ppm lysine, 5 ppm cystine, 0.05 ppm glutamic acid, and 15% (w/v) sucrose (14Saini J.P. Dube S.D. Maydica. 1986; 31: 227-232Google Scholar). More than 80% of the pollen germinated, and the length of the pollen tube was about 0.5 and 4 times the diameter of the pollen after 10 and 30 min, respectively. Preparation of an Anther Wall Fraction and a Microspore Fraction— Each anther of developmental stage 3 was placed in a drop of solution containing 0.05 m sodium acetate, pH 5.2, and 0.4 m sucrose on a dish and sliced open longitudinally with a scalpel under a dissecting microscope. The microspores were gently scraped from the anther wall. The microspores and the anther wall, which still retained about 10% of the original microspores, were collected separately. Each of the two fractions from 10 anthers were combined and subjected to RNA extraction. Preparation of a Total Pollen Fraction, a Pollen-released Protein Fraction, an Interior Fraction, and a Pollen Shell Fraction—Fresh pollen was homogenized rigorously in 0.01 m sodium acetate, pH 5.2, with a mortar and pestle to yield a total protein fraction. A sample of 20 mg of fresh pollen was placed in a 1.5-ml microcentrifuge tube containing 1 ml of 0.01 m sodium acetate, pH 5.2 or the germination medium described in a preceding paragraph. The preparation was shaken gently for 10, 20, or 60 min and centrifuged at 8,000 × g for 3 min. The supernatant was collected as the pollen-released protein fraction. The pellet was homogenized gently in 0.2 ml of 0.01 m sodium acetate, pH 5.2 with a small mortar and pestle, and the homogenate was referred to as the pollen interior fraction. The homogenate was put onto a sucrose gradient containing, from top to bottom, 0.5 ml each of 1, 1.5, and 1.9 m sucrose in 0.05 m HEPE-NaOH, pH 7.5, and centrifuged at 12,000 × g for 30 min. The interface materials between the 1 and 1.5 m sucrose solution were collected. The sample was mixed with three volumes of 0.05 m HEPE-NaOH, pH 7.5, and centrifuged at 8,000 × g for 3 min. The pellet was resuspended with a small volume of 0.05 m HEPE-NaOH, pH 7.5, and was referred to as the pollen shell fraction. Extraction of a Coat Fraction by Diethyl Ether from Pollen and Partition of Its Proteins into an Aqueous Medium for Enzymatic Studies—Freshly collected pollen was mixed with diethyl ether (1 g of pollen/10 ml of ether) for 1 min in a capped tube by repeated inversions. The ether layer was collected after centrifugation for 10 min at 8,000 × g. The ether preparation was reduced to 1 ml under a stream of nitrogen and used as the coat fraction for SDS-PAGE. Also, the proteins in this coat fraction were partitioned into an equal volume of 0.05 m sodium acetate, pH 5.2 for fast protein liquid chromatography (FPLC). 1The abbreviations used are: FPLC, fast protein liquid chromatography; ER, endoplasmic reticulum; EST, expressed sequence tag; 4-NPG, p-nitrophenyl β-d-glucopyranoside; RT, reverse transcriptase; UTR, untranslated region; PIPES, 1,4-piperazinediethanesulfonic acid; PBS, phosphate-buffered saline; RACE, rapid amplification of cDNA ends. Cation Exchange FPLC—The procedure was as described earlier (8Bih F.Y. Wu S.S.H. Ratnayake C. Walling L.L. Nothnagel E.A. Huang A.H.C. J. Biol. Chem. 1999; 274: 22884-22894Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). All solutions contained 0.05 m sodium acetate, pH 5.2. The aqueous pollen coat sample (∼30-μg proteins in 3 ml) described in the preceding paragraph was filtered through a 0.2-μm syringe filter and applied to a pre-equilibrated Mono S HR 5/5 FPLC column (Amersham Biosciences). Solutions of 5 ml of 0 m NaCl, 6 ml of 0.25 m NaCl, 15 ml of a linear gradient of 0.25–0.75 m NaCl, 5 ml of 1 m NaCl, and 5 ml of 2 m NaCl were applied successively to the column. Chromatographic fractions of 0.5 ml each were collected and analyzed for protein constituents by SDS-PAGE and β-glucanase and xylanase activities. The fractions containing the purified 70- and 35-kDa proteins had the peak activities of β-glucanase and xylanase, respectively and were retained for additional analyses of enzyme activities. Enzyme Activity Assay—β-glucanase activity was measured by monitoring the appearance of reducing ends from substrates at time intervals. Laminarin from Laminarin digitata (Sigma) was used as the substrate. The β-glucanase reaction mixture of 0.3 ml contained the purified 70-kDa protein fraction and 0.3 mg laminarin in a 0.05 m buffer, which included sodium acetate, pH 4.0, 5.0, and 5.5; succinate-NaOH, pH 5.5, and 6; phosphate-NaOH, pH 6, 7, 8, and 8.5; CHES-NaOH, pH 8.5, 9, and 10. The reaction was allowed to proceed at 30 °C and terminated by the addition of 0.9 ml of p-hydroxybenzoic acid hydrazide reagent and then heating (15Lever M. Anal. Biochem. 1977; 81: 21-27Crossref PubMed Scopus (163) Google Scholar). After the mixture was cooled, the absorbance of the reaction mixture was read at 410 nm with a spectrophotometer. The enzyme activity was monitored at four time intervals within a 6-h period to ensure linearity of the reaction. The activities of the purified coat β-glucanase fraction, the purified coat xylanase fraction, and Aspergillus niger β-glucanase (EC 3.2.1.4, from Sigma) on various substrates (xylan, lichenan, carboxymethylcellulose, and polygalacturonic acid; Sigma) in 0.05 m sodium acetate, pH 5.2, were assayed as described for laminarin. Activities of the various enzyme preparations on 0.15 mg of p-nitrophenyl β-d-glucopyranoside (4-NPG; Sigma) were assayed in 0.3 ml of 0.05 m sodium acetate, pH 5.2, at 30 °C. The reaction was terminated by the addition of 0.6 ml of 4% (w/v) Na2CO3 (16Biely P. Vrsanska M. Kratky Z. Eur. J. Biochem. 1980; 108: 313-321Crossref PubMed Scopus (103) Google Scholar), and the absorbance of the reaction mixture was read at 410 nm with a spectrophotometer. SDS-PAGE of Proteins for Separation, Antibody Preparation, and Peptide Microsequencing—Acrylamide (12.5%, w/v) SDS-PAGE was performed as described (5Wu S.S.H. Platt K.A. Ratnayake C. Wang T.W. Ting J.T.L. Huang A.H.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12711-12716Crossref PubMed Scopus (96) Google Scholar). The proteins in the gels were stained with Coomassie Blue. For antibody preparation, gel slices containing the 10- and 17-kDa proteins from the pollen-released and interior fractions, respectively, were cut from the gel and used to produce antibodies in chicken (5Wu S.S.H. Platt K.A. Ratnayake C. Wang T.W. Ting J.T.L. Huang A.H.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12711-12716Crossref PubMed Scopus (96) Google Scholar). For microsequencing, fractionated proteins in a gel were transferred to a polyvinyldene fluoride membrane (Millipore Corp., Bedford, MA) (9Wu S.S.H. Suen D.F. Chang H.C. Huang A.H.C. J. Biol. Chem. 2002; 277: 49055-49064Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) and sequenced at their N termini at the Genomic Institute at the University of California, Riverside (Riverside, CA). Alternatively, the proteins in the gel were subjected to trypsin digestion (for the 14-, 25-, and 70-kDa proteins) and their fragments sequenced at the Iowa State University Protein Facility (Ames, IA). The obtained sequences of the 10-, 14-, 17-, 20-, 23-, 25-, 35-, 38-, 47-, and 70-kDa proteins were TTPLTFQVGKGSKPG, DFDEPGHLAP, KKKRAAESGEAAEAK, ATLAEICRGTAFPDI, FVVTGRIYCDNCRAG, LPAQSPTLK, GPPKVPPGKNITATY, RFIGGVGDDY, EKEESKGIDAKA, and YFVGSVLSGG, respectively. Analysis of Nucleotide and Amino Acid Sequences—We used the NCBI (www.ncbi.nlm.gov) and TIGR (www.tigr.org) data bases to search for EST nucleotide sequences or protein sequences. The GCG Program (gcg.ucr.edu) was used to compare nucleotide sequences or amino acid sequences, to construct phylogenetic trees and to translate nucleotide sequences into amino acid sequences. Subcellular locations of proteins were analyzed by use of the PSORT Program in ExPASy (us.expasy.org). RNA Extraction and RT-PCR—Total RNA was extracted (17Kim H.U. Hsieh K. Ratnayake C. Huang A.H.C. J. Biol. Chem. 2002; 277: 22677-22684Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar) from maize anthers of different developmental stages, mature pollen and germinated (after 15–30 min) pollen. A sample of 1 μg of total RNA was treated with RNase-free DNase and then used to synthesize first-strand cDNA with an oligo(dT)15 primer (18Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). The cDNA was used as a template for RT-PCR with a pair of primers. For the gene encoding the pollen-coat 70-kDa β-glucanase, the 5′ primer (5′-CAGATCGAGAGGGCCAACGC-3′) and 3′ primer (5′-CTTGACGAGGCGGGGGTCC-3′) were designed from the coding region of the new maize gene ZmGLA3 (AY344632), which is different from those of the two known maize β-glucanases (to be described in Fig. 3). For the gene encoding the pollen-coat xylanase, the 5′ primer (5′-GGGAGGCATGACCGCTTACT-3′) and 3′ primer (5′-CTTGGTGACCGCGTTGCCG-3′) were designed from the coding region of the maize gene ZmXYN1 (AF149016). For the gene encoding the pollen-released 10-kDa protein, the 5′ primer (5′-CACCAACAATGGCCTCCAGG-3′) and 3′ primer (5′-GCTGAGTGTGTCTATAGCGTG-3′) were designed from the coding region of a maize EST clone, AY111779. For the gene encoding the pollen-released 14-kDa protein, the 5′ primer (5′-TGATGTTGCTGGTTCCAACTT-3′) and 3′ primer (5′-TGCTTAGTAATTGGAGTCTGTT-3′) were designed from the 3′-UTR of a maize profilin gene, ZmPRO1, X73279. For the gene encoding the pollen-wall 17-kDa protein, the 5′ primer (5′-GCCCCTTGAAATGAATGAAC-3′) and 3′ primer (5′-ACATTTGCGTGACATTACATTT-3′) were designed from the 3′-UTR of a maize EST clone, TC148758. For the gene encoding the pollen-released 20-kDa protein, the 5′ primer (5′-GATCTCCTTCTCCACGGACG-3′) and 3′ primer (5′-ATTCCACCTCAATTCGCCTCC-3′) were designed from the 3′-UTR of a maize EST clone, TC132556. For the gene encoding the pollen-released 23-kDa protein, the 5′ primer (5′-ATGCGCGCATGCATGCTAGC-3′) and 3′ primer (5′-AGATGGTCAAGAATTGAATTCAC-3′) were designed from the 3′-UTR of a maize EST clone, TC130551. For the gene encoding the pollen-released 35-kDa protein, the 5′ primer (5′-GTGCTGACAATACTTTAAGCCG-3′) and 3′ primer (5′-ACAACACAACATTCATGGATCC-3′) were designed from the 3′-UTR of a maize EST clone, TC148755. For the gene encoding the pollen-released 47-kDa protein, the 5′ primer (5′-CGACTGACCCATCTCTCTA-3′) and 3′ primer (5′-GAGAGAGAGAGACAAGAGGT-3′) were designed from the 3′-UTR of a maize EST clone, X57627. The PCR product was analyzed on a 1.2% agarose gel and purified with the use of the QIAGENE Gel extraction kit (Qiagene, Valencia, CA). The purified PCR product was 32P-labeled with the use of Prime-a-Gene Labeling system (Promega Corp., Madison, WI). The 32P-labeled fragment was used as a probe for RNA blot hybridization. RNA Blot Hybridization—Each sample of 30 μg of total RNA was fractionated with the use of a 1.2% formaldehyde gel by electrophoresis. The gel was equilibrated in 10× SSC, pH 7.0 for 20 min. After equilibration, the RNAs were blotted onto a Hybond-N membrane (Amersham Biosciences). The RNA-blotted membrane was prehybridized at 65 °C in potassium phosphate, pH 7.2, 7% SDS, 1% bovine serum albumin, and 0.01 m EDTA, pH 8.0 for 4 h; hybridized with 32P-labeled probes (preceding paragraph) overnight; and then washed with 2× SSC, 0.1% SDS for 20 min; 1× SSC, 0.1% SDS for 20 min; and 0.1× SSC, 0.1% SDS for 20 min, all at 65 °C. RT-PCR Analysis of ZmGLA1, ZmGLA2, and ZmGLA3 Transcripts in Maize Anthers, Mature Pollen, and Germinated Pollen—Total RNA from maize anthers of different developmental stages, mature pollen or germinated pollen was extracted and used to synthesize first-strand cDNA, as described in a preceding section. The cDNA was used as templates for PCR with a pair of gene-specific primers. For ZmGLA1, the 5′ primer (5′-GTGTGAGGCGCTCTGATGG-3′) and 3′ primer (5′-ACACGGCTAAATAGGGTATGG-3′) were designed from the 3′-UTR of a maize β-glucanase ExoI EST clone, AY103742 (gDNA, AF225411). For ZmGLA2, the 5′ primer (5′-AGATGTACCAGAACTAGAAGAA-3′) and 3′ primer (5′-AAGAATGAGATGGCTCATATGT-3′) were designed from the 3′-UTR of a maize β-glucanase (ExoII) EST clone, AF064707. For ZmGLA3, the 5′ primer (5′-GCCGCGAGTCACGATTAGC-3′) and 3′ primer (5′-CAATCTTTTTTTAACTACACCTAC-3′) were designed from the 3′-UTR of our newly registered maize gene (GenBank™ AY344632). For a maize actin gene, the 5′ primer (5′-GGTTACTCCTTCACCACGAC-3′) and 3′ primer (5′-CAGACACTGTACTTCCTCTCAG-3′) were designed from the coding region of a maize actin gene (GenBank™, Maz56, U60514). The PCR fragments were analyzed with 1.2% agarose gels. 5′-Rapid Amplification of cDNA Ends (5′-RACE)—It was performed with the use of a 5′-RACE system (Invitrogen) according to the provided instructions. The first-strand cDNA was synthesized with a primer, 5′-GTTGTGGGGGAAGAT-3′. Primary and nested PCR products were synthesized with the primers, 5′-GGCGTTGTAGACGTCGTTGTT-3′ and 5′-CCGTGCACGGCGTCGATGC-3′, respectively. The above three primers were designed on the basis of a maize gDNA clone, BZ402366, which is highly similar to the 5′-terminus of a rice EST clone, TC81322. The sequence of the new maize gene, termed ZmGLA3 (registered as GenBank™ AY344632), was assembled from the sequenced 5′-RACE product, maize EST TC163747 and maize gDNA BZ402366, BZ533772, BZ530441, and BZ533777. Microscopy—Samples of pollen before and after protein release were observed under a Zeiss Axiophot microscope and then photographed. For immunofluorescence microscopy, germinated pollen grains having different tube lengths in the germination medium were fixed by mixing with 1 volume of 2× fixation solution (1×: 4% (w/v) paraformaldehyde, 50 mm PIPES buffer, pH 6.9, 2 mm MgSO4), and 15% (w/v) sucrose). The preparation was placed at room temperature for 1 h. The pollen grains were transferred to a 1× fixation solution (omitting sucrose) at room temperature for 1 h. After fixation, the pollen grains were washed with PBS (10 mm phosphate-buffered saline, pH 7.4, 138 mm NaCl, 2.7 mm KCl) three times for 15 min each. The pollen grains were blocked with 3% (w/v) nonfat dry milk in PBS at room temperature for 2 h. The blocked pollen grains were allowed to react with chicken antibodies against the maize pollen 10- or 17-kDa protein (1:100 dilution) in PBS containing 1% (w/v) nonfat dry milk at 4 °C overnight. After being washed with PBST (PBS containing 0.05% (v/v) Triton X-100) 3 times for 10 min each, the pollen grains were incubated with Cyanine 3-conjugated donkey secondary antibodies against chicken IgG or IgY (Jackson Immuno Research Lab., West Grove, PA) at room temperature for 2–3 h. After being washed with PBST three times for 10 min each, the pollen grains were mounted in an antifade reagent from SlowFade Antifade Kit (Molecular Probes, Eugene, OR) and viewed under a Leica SP2 UV confocal microscope. Mature Maize Pollen Was Separated into Fractions of Distinct Structures or Origins—Mature pollen was separated into four distinct fractions, the coat, a pollen-released protein fraction, the interior, and the shell. These fractions were analyzed for their protein constituents by SDS-PAGE. A coat fraction was obtained by washing the mature pollen with diethyl ether. SDS-PAGE revealed three visible protein bands, which represented only a very small proportion of the total pollen proteins (Fig. 1A). N-terminal sequencing and FPLC revealed that the 70- and 35-kDa protein bands each represented one protein (to be described) and that the 25-kDa protein band contained at least two fragments of different proteins via a search of GenBank™ protein data bases. These fragments were not studied further. Fractions of pollen-released proteins and pollen interior proteins were obtained by the following procedure. Mature pollen was shaken gently in a liquid medium of 10 mm sodium acetate, pH 5.2 for 20 min, during which most of the pollen (about 95%) did not burst (Fig. 1, B and C). The proteins released from and those retained in the pollen were analyzed by SDS-PAGE. The released proteins in the designated pollen-released protein fraction represented about 10% of the total pollen proteins and were separated into several sharp protein bands on the gel (Fig. 1A). The proteins retained in the pollen in the designated pollen interior fraction were separated into many bands. The pollen-released and interior proteins resolved in the gel were mostly non-overlapping, which indicates the selectivity of the separation procedure. After the almost complete liberation of the pollen-released proteins, the pollen still retained its interior density as observed by light microscopy (Fig. 1, B and C). Both the pollen-released and interior fractions contained some of the coat proteins, which are not visible from the stained SDS-PAGE gel because of their relatively minute amounts (Fig. 1A) but were revealed by immunoblotting with antibodies against the 35-kDa proteins (data not shown) A pollen shell fraction, representing the pollen after the pollen-released and interior proteins had been removed, was obtained by gently grinding the interior fraction and subjecting the ground materials to sucrose gradient centrifugation. A clean fraction of individual pollen shells was ob" @default.
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