Maize Pollen Coat Xylanase Facilitates Pollen Tube Penetration into Silk during Sexual Reproduction*

Cell wall hydrolases are well documented to be present on pollen, but their roles on the stigma during sexual reproduction have not been previously demonstrated. We explored the function of the tapetum-synthesized xylanase, ZmXYN1, on maize (Zea mays L.) pollen. Transgenic lines (xyl-less) containing little or no xylanase in the pollen coat were generated with use of an antisense construct of the xylanase gene-coding region driven by the XYN1 gene promoter. Xyl-less and wild-type plants had similar vegetative growth. Electron microscopy revealed no appreciable morphological difference in anther cells and pollen between xyl-less lines and the wild type, whereas immunofluorescence microscopy and biochemical analyses indicated an absence of xylanase on xyl-less pollen. Xyl-less pollen germinated as efficiently as wild-type pollen in vitro in a liquid medium but less so on gel media of increasing solidity or on silk, which is indicative of partial impaired water uptake. Once germinated in vitro or on silk, the xyl-less and wild-type pollen tubes elongated at comparable rates. Tubes of germinated xyl-less pollen on silk did not penetrate into the silk as efficiently as tubes of wild-type pollen, and this lower efficiency could be overcome by the addition of xylanase to the silk. For wild-type pollen, coat xylanase activity on oat spelled xylan in vitro and tube penetration into silk were inhibited by xylose but not glucose. The overall findings indicate that maize pollen coat xylanase facilitates pollen tube penetration into silk via enzymatic xylan hydrolysis.

Sexual reproduction in plants is the union of male gametes in pollen and eggs in the female carpel of flowers. A pollen grain lands and germinates on the carpel surface, termed the stigma, and produces a tube that penetrates the stigma into the carpel. The tube carrying male gametes elongates toward the ovary and discharges them to the ovule, where one of the two gametes fertilizes the egg (1)(2)(3).
Initial contact during sexual reproduction involves two successive steps, namely, germination of pollen on the stigma and penetration of the pollen tube through the stigma into the carpel. The former step is more critical in species with dry stigma, such as maize and those of Brassicaceae (4). Dry stigma discour-ages microbial infection, but the pollen has to draw water from the carpel interior for germination. The mechanism for pollen to absorb water from the carpel interior depends on the species. In self-incompatible species, such as those in the Brassica genus, water uptake from the carpel relies on a haplotype-specific signal transduction system, which permits water uptake into compatible pollen (5,6). In self-compatible species, such as maize and most Arabidopsis species, water uptake does not require this signal transduction system. Regardless, for pollen to obtain water from the carpel, its coat has to come into physical contact with the stigma to establish water flow (7). In addition to its ability to absorb water and germinate on the stigma, pollen placed in a liquid or on a semisolid medium readily takes up water and germinate (8,9).
After the pollen tube has protruded from the grain, it needs to penetrate the stigma into the carpel. The mechanism of this step is unknown, in part, because of the difficulty in studying the in vivo process. The stigma wall is overlaid with an incomplete layer of cutin, and thus the crucial physical barrier is the wall alone (7). Wall hydrolases and wall-modulating proteins are known to be present on or rapidly secreted from pollen. Their presence was observed by in situ staining (10) and by chemical analyses of extracts of pollen coat, subfractions of the pollen wall, or rapidly secreted materials from pollen (11)(12)(13)(14)(15)(16)(17)(18). These proteins could be remnants of lyzed tapetum cells stuck onto the pollen having no further function. Alternatively, they could be components specifically synthesized for hydrolysis of the stigma wall for tube entry or hydrolysis and modulation of the tube track wall inside the carpel for advancement of the pollen tube.
Pollen wall hydrolases and wall-modulating proteins have been best studied with maize. Maize is monoecious and selfcompatible, and its flowers are large and produce abundant pollen. Maize pollen surface crest (19) can be separated into two biochemically distinct, morphological parts: the wall and the coat external to it (17).
Maize pollen wall and its transiently associated proteins include wall hydrolases and modulating proteins (11)(12)(13)(14)(15)(16)(17)(18). These proteins are synthesized in the pollen interior and secreted to the wall and exterior before, during, or immediately after germination. They include polygalacturonase and several wall-modulating proteins, which consist of expansin, profilin, cation-binding protein, pollen allergen (trypsin inhibitor), extensin, and other potential wall modulators. The mRNAs of these proteins in the microspores (immature pollen) and mature pollen appear late during anther development, and their levels persist or increase after germination (17). Thus, these wall hydrolases and modulating proteins likely exert a role after the pollen tube has penetrated the stigma and may hydrolyze and modulate the cell wall of the cells forming the pollen tube track in the carpel for advancement of the pollen tube toward the ovary.
Maize pollen coat, which is located on top of the pollen wall, contains proteins and lipids. These coat molecules are largely if not exclusively synthesized in the adjacent tapetum, which is a one-celled layer enclosing the anther locule where pollen mature (20). The coat proteins consist of only a few molecular species, the predominant one being a xylanase (EC 3.2.1.8) (14). Their mRNAs are present exclusively in the tapetum and appear earlier than those of the pollen-synthesized wall proteins (preceding paragraph) during anther development. The xylanase is synthesized as a larger precursor of 60 kDa in the tapetum and is processed at both the N and C termini to the active 35-kDa enzyme (21). These coat proteins may be present fortuitously as remnants after death of the tapetum or, especially the predominant xylanase, play a vital and yet unknown physiological role in the initiation of sexual reproduction.
Maize and other cereals have a Type II cell wall, which contains predominantly hemicellulose and cellulose (22). The hemicellulose backbone consists of a polymer of xylose (xylan) with short branches of arabinose and glucuronic acid. The xylanase in the maize pollen coat has been purified to homogeneity, and its in vitro activity is that of an endoxylanase that can act specifically on xylan (14,17). Thus, the maize pollen coat xylanase may act on the hemicellulose in the stigma wall, generating an opening for entry of the pollen tube into the carpel.
We explored this possible role of the maize pollen coat xylanase. We used an antisense approach to generate lines with normal vegetative growth but little or no xylanase on pollen. We then examined the ability of xylanase-less pollen to germinate in vitro and in vivo and their tubes to penetrate the stigma into the carpel. The overall results show that the coat xylanase facilitates pollen tube penetration into the carpel via enzymatic xylan hydrolysis.

EXPERIMENTAL PROCEDURES
Plant Materials-Anthers and pollen were collected from maize (Zea mays L., B73) plants grown in a greenhouse at 28°C with 14-h/10-h day/night cycle. Anthers of five developmental stages were selected on the basis of the following criteria (14). 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. 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.
Cloning and Sequencing of Xyl Antisense Construct-A 2.0-kb ZmXYN1 fragment was amplified from a genomic clone (14) by PCR with 5Ј(5Ј-GCGGCCGCTGAAGATAACATAATTAA-GATATCG-3Ј) and 3Ј (5Ј-GGATCCCTTGGTGACCGCGT-TGCCG-3Ј) primers; the underlined sequences represent those recognized by the applied restriction enzymes. This fragment contains a 1.2-kb ZmXYN1 5Ј promoter sequence plus the 5Ј-untranslated region and a 744-bp sequence (preXYN1) encoding the N-terminal 248 residues of the 60-kDa prexylanase; the 248-residue polypeptide encoded by the latter sequence would be removed in in vivo processing of the prexylanase to the mature 35-kDa xylanase. A 931-bp ORF 2 encoding the mature 35-kDa xylanase was amplified from the same genomic clone by PCR with 5Ј (5Ј-GGATCCCTCGGCCTCC-CGGCATACG-3Ј) and 3Ј (5Ј-GGATCCCTTACGGTGAAC-TTAAAGTCGC-3Ј) primers. The 2.0-and 931-bp fragments were cloned into pBluescript II SK(ϩ) (pBSK) vector and pGEM-T, respectively. A 0.3-kb nopaline synthase terminator (NOS-ter) was cut from pBI 101.1 with use of SacI and EcoRI and then cloned into pUC19. The NOS-ter fragment was cut from this pU19 with BamHI and EcoRI and inserted into pBSK containing (ZmXYN1 promoter ϩ N-preXYN). The ORF of ZmXYN1 encoding the 35-kDa xylanase was cut from pGEM-T containing the insert with use of BamHI and inserted into pBSK containing ZmXYN1 promoter ϩ N-preXYN ϩ NOS-ter at the position between the N-preXYN and NOS-ter sequences. The antisense orientation and the sequence of the ORF of ZmXYN1 were confirmed with results from restriction enzyme digestions and sequencing, respectively.
Establishment of Xyl Antisense Transgenic Lines-Immature embryos of maize (Z. mays L., B73) were bombarded with the plasmid containing the above-mentioned antisense XYN1 construct and a plasmid containing a bar gene (23) with use of a gene gun. After bombardment, calli were initiated from the embryos in a selection medium for bialaphos resistance. A portion of each selected callus was used to detect the inserted antisense XYN in the genome by PCR. Calli containing the proper antisense XYN1 construct were regenerated into plantlets on MSS media (MS medium plus vitamins, 3% sucrose, and 0.3% gelrite but minus hormones) in Petri dishes (25 ϫ 100 mm) at 25°C in 80 -100 E/m 2 /s light (16/8-h photoperiod). The bombardment and regeneration of plantlets were performed at the Plant Transformation Facility at Iowa State University. When the leaves and roots of a plantlet reached about 4 -6 cm long, the plantlet was transferred to soil and grown in a greenhouse at 28°C with a 14/10-h photoperiod. Regenerated transgenic plants (T 0 ) were pollinated with wild-type (inbred B73) pollen, and the seeds so produced were termed T 1 . T 1 plants grown from T 1 seeds produced flowers, and the pollen was subjected to immunoblot analyses for XYN1 (a following section). Individual T 1 plants that produced pollen with little or no XYN1 were retained and selfed, and the seeds produced were termed T 2 . Plants grown from T 2 seeds were used for experiments.
PCR Analysis of Transgenic Lines-Genomic DNA was extracted from transformed calli by cetyltrimethylammonium bromide (24). PCR analysis involved use of 5Ј (5Ј-GCGGCC-GCTGAAGATAACATAATTAAGATATCG-3Ј) and 3Ј (5Ј-CGAGAACGAGATGAAGTGGTAC-3Ј) primers and yielded a 3.0-kb fragment, representing the promoter and the coding regions of XYN1 (shown in Fig. 1). The PCR products were analyzed by electrophoresis in a 1% agarose gel with ethidium bromide staining and photographed under UV. Calli whose DNA gave the 3.0-kb fragment represented transformed lines that had the correct inserted antisense XYN1 construct in the genome. These calli were regenerated into mature plants (T 0 ).
Genomic DNA Blot Hybridization of XYN1-Genomic DNA was extracted from leaves by cetyltrimethylammonium bromide (24). Each hybridization reaction mixture contained 10 g of gDNA and one restriction enzyme (EcoRI or HindIII). After digestion, the DNA fragments were separated with the use of a 0.8% agarose gel. The gel was placed in 0.25 M HCl for 20 min, rinsed with water twice, shaken in 1.5 M NaCl and 0.5 M NaOH for 1 h, and equilibrated in 1 M Tris-HCl, pH 8.0, and 1.5 M NaCl for 1 h. The DNA fragments were blotted onto a Hybond-N membrane (Amersham Biosciences). The membrane was pretreated at 65°C in 0.5 M sodium phosphate, pH 7.2, 7% SDS, 1% bovine serum albumin, and 0.1 M EDTA for 4 h, hybridized with 32 P-labeled gene-specific probe overnight, and then washed with 2ϫ SSC, 0.1% SDS for 20 min, 1ϫ SSC, 0.1% SDS for 20 min, and 0.1ϫ SSC, 0.1% SDS for 20 min, all at 65°C. The 32 P-labeled gene-specific probe was prepared as follows. The plasmid contained the antisense construct was digested with BamHI and EcoRI (Fig. 1A). After digestion, the reaction mixture was run on a 1% gel. The gel containing the expected 494-bp fragment was cut, and the fragment was extracted and purified with the use of QIAquick gel extraction kit (Qiagen, Valencia, CA) and then 32 P labeled with the use of Prime-a-Gene labeling system (Promega, Madison, WI). Un-incorporated, labeled nucleotide was removed with use of a Micro-Spin TM G-50 column (Amersham Biosciences). The purified probe was used in the hybridization reaction.
Preparation of Total Pollen Protein and Pollen Coat Fraction-A total pollen-protein fraction was obtained as follows. A sample of 5.5 mg of mature pollen was ground in 100 l of 2ϫ SDS-PAGE sample buffer (1ϫ: 0.0625 M Tris-HCl, pH 6.8, 2% SDS, 5% ␤-mercaptoethanol, 0.002% bromphenol blue, and 10% glycerol) in a microcentrifuge tube with a mini-pestle, and the homogenate was boiled for 20 min. A pollen coat fraction was obtained as follows. One gram of pollen was mixed with 10 ml of diethyl ether for 3 min in a capped tube by repeated inversions. The tube was centrifuged for 10 min at 8,000 ϫ g, and the ether layer was collected. The volume of the ether preparation was reduced to 500 l under a stream of nitrogen. The ether preparation was used directly for lipid analyses by TLC or partitioned with 1 volume of 50 mM sodium acetate, pH 5.0. The aqueous sodium acetate fraction (lower layer) was collected and used as the pollen coat protein fraction.
Detection of Pollen Coat Xylanase and ␤-Glucanase by SDS-PAGE and Immunoblot Analyses and of Lipids by TLC-All procedures followed those described earlier (14). The total proteins of pollen from wild-type and transgenic plants were subjected to 12.5% (w/v) SDS-PAGE. After electrophoresis, the gel was stained with Coomassie Blue and then destained or subjected to immunoblot analysis as follows. The proteins on the gel were transferred to a nitrocellulose membrane. Rabbit antibodies were raised against the purified recombinant mature 35-kDa XYN1 (14). The antibody preparation recognized the pollen coat mature 35-kDa XYN1 and also a 55-kDa pollen interior protein, whose identity is unknown (21). The latter immunodetected protein was used as a control of equal loading of total pollen proteins from different transgenic maize lines into a gel for comparison. Serial-diluted total pollen-protein fraction of the wild type (1ϫ, 2ϫ, 4ϫ, and 8ϫ) and serial-increased total pollen-protein fractions individual transgenic lines (1ϫ and 2ϫ) were used to estimate the loss of xylanase in the transgenic lines. Transgenic lines producing pollen coat xylanase with less than 20% of that in wild-type were used; they are called the xyl-less line or pollen.
The above total pollen protein sample after SDS-PAGE was also used for immunoblot analyses of the coat ␤-glucanase. Rabbit antibodies were raised against a synthetic polypeptide of DKDDLAKSAEGYE, which was present in the maize pollen coat ␤-glucanase but absent in all other known maize ␤-glucanases (17). Coat lipids were applied to a TLC plate (silica gel 60 A, Whatman Inc., Clifton, NJ). The plate was developed in hexane:diethyl ether:acetic acid (95:5:2, v/v/v) and stained in an iodine chamber for 24 h.
Pollen Germination in Vitro-Old pollen and anthers on tassels of plants were removed in the afternoon a day before pollen collection. Fresh pollen was collected from each plant in the morning by shaking the tassel in a plastic bag. For in vitro germination, a sample of 5 mg of pollen was placed in 2 ml of liquid germination medium (100 ppm Ca(NO 3 ) 2 , 10 ppm H 3 BO 3 , 37.5 ppm lysine, 5 ppm cysteine, 0.05 ppm glutamic acid, and 15% (w/v) sucrose (8)) or on solid germination media. Solid media had the same composition as that of the liquid medium but with the addition of 0.7, 1.4, or 2.1% (w/v) of phytagel (Sigma catalog number P-8169). Each gel medium was placed in a 100 ϫ 15-mm Petri dish, which was then air-dried under a laminar hood for 2 h to remove water on the medium surface. The dish was sealed with a parafilm and stored at 4°C. It was warmed to room temperature immediately before use. Pollen was spread on the gel, and the dish was covered to keep moisture. The pollen was allowed to germinate at 25°C for periods of time described under "Results." Germinated and non-germinated pollen grains were observed with a Leica DMLB microscope and counted.
Pollen Germination on and Tube Penetration into Silk-Fresh silks on each wild-type plant were covered with a paper bag after the silks started to emerge. All silks used in one experiment were obtained from the same plant. When the silks that had emerged from the cob were 8 cm long, their tips turned slightly pink. These 8-cm silks were cut, and their lower ϳ1-cm portions were placed in a cylindrical vial (5 cm tall and 1.3 cm internal diameter) containing 2 ml of water. Pollen grains were dusted onto the top ϳ5-cm portions of 16 silks to saturation. The silks were then shaken gently to remove pollen grains that did not adhere tightly. The vial including silks was placed at the center of a 1000-ml beaker, which contained 100 ml of water. Prior to this placement, the beaker was sealed with a transparent plastic for 24 h to generate a relative humidity close to saturation (ϳ100%). Samples were quickly placed in and removed from the beaker, which was then resealed. At time intervals, silks were removed from the vial, and their upper portions were cut into three segments of 1 cm each and fixed on a microscopy glass slid with use of a fixation solution (2.5% glutaraldehyde and 0.05% Titon X-100 in PBS (10 mM phosphate-buffered saline, pH 7.4, 138 mM NaCl, and 2.7 mM KCl)) for 1 h. The fixed silk segments were observed directly or after treatment with 0.1% aniline blue for 1 h and photographed with a Leica DMLB microscope. All pollen grains (200ϳ400) from three silk segments on one slid at each time interval were observed and counted. Pollen tubes penetrated into or grew along the surface of the silks. These two types of pollen tubes were determined by observing their fluorescence (excitation at 530 -560 nm and emission detected at 570 -650 nm) with use of a microscope and continuous focus adjustments (shown in Fig. 6). The germination ratio of a pollen sample is the number of germinated pollen divided by the number of total pollen on three silk segments from one silk on a slid. The penetration efficiency of a pollen sample is the number of pollen with tubes penetrating into silks divided by the number of germinated pollen on the same slid.
Experiments described in the preceding paragraph were also performed to test the effects of xylanase, BSA, xylose, and glucose on pollen germination and tube penetration. Viability of pollen grains was severely compromised after they had been treated with any tested solution for 5 min. Therefore, the silks were treated instead. The top 6-to 7-cm portions of the 8-cm silk segments (preceding paragraph) before being dusted with pollen were first immersed in a solution of 0.4 g/l of BSA, 0.4 g/l of a Thermomyces lanuginosus xylanase (Sigma catalog number X-2753), 0.1 M xylose or glucose, or a blank solution for 5 min and then dried in air for 10 min. The silk segments were then treated as described in the preceding paragraph.
Measurement of Pollen Tube Length-Pollen was germinated in a liquid or 2.1% phytogel medium as described in the preceding paragraph. When the pollen germination ratio was about 10% (after 5 min in liquid and 20 min in gel medium), 10 or more germinated pollen grains were randomly chosen, and the lengths of tubes were estimated at time intervals. For convenience of estimation by microscopy, the unit of tube length was the diameter of a pollen grain (ϳ70 m).
Statistical Analyses-S.E. values shown in all figures that illustrate percents of germination or tube penetration were calculated from the following equation, in which p (percent germination) ϭ x/n, n ϭ the number of pollen grains used, and x ϭ the number of pollen grains germinated. The n was about 200 for pollen germination in vitro and about 200 -400 for pollen germination on or penetration into silk. The S.E. values of tube elongation (Fig. 4) were calculated from the following equation, in which x ϭ fold of pollen diameter, x ϭ average of 10 randomly picked tubes, and x i ϭ fold of each sample. Z test was used to test whether the difference between two data points was significant.
Measurement of Xylanase Activity by Viscosity Assay-All procedures followed those described earlier (14). An amount of 200 mg of oat spelled xylan (Sigma catalog number X-0627) was mixed with 2 ml of water in a capped tube with continuous, slow inversion. The mixture was centrifuged at 2,000 ϫ g for 10 min. The supernatant was used as the substrate for xylanase. Each enzyme assay contained 1 ml of oat spelled xylan, 20 l of pollen coat fraction (about 6.5 g proteins), or 0.01 unit of T. lanuginosus xylanase, sodium acetate (0.15 M final concentration), and water to a final volume of 1.2 ml. When desired, the assay mixture also contained 0.1 M xylose or glucose. The assay mixture was placed at 30°C. At time intervals, a volume of 0.15 ml of the assay mixture was used for viscosity testing. The volume was taken into a 0.2-ml viscosity pipette and then allowed to flow down vertically. The flow time for a 0.15-ml reaction mixture without enzyme was 6.3 s.
Electron Microscopy-For transmission EM, all steps were as described previously (26). Anthers at different stages from the wild-type and a xyl-less plant were cut and fixed in a fixation solution (2.5% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.0) for 2 h. The samples were washed with 0.1 M sodium phosphate buffer, pH 7.0, post-fixed with 1% of OsO 4 in 0.1 M sodium phosphate buffer, pH 7.0, for 2 h, washed with the buffer without osmium, then dehydrated, embedded, sectioned, and post-stained with uranyl acetate and lead citrate. For scanning EM, pollen was fixed with 2.5% glutaraldehyde solution overnight at 4°C, washed with 0.1 M sodium phosphate buffer, pH 7.0, and post-fixed in 1% of OsO 4 for 2 h at 4°C. The samples were then washed with the buffer and dehydrated in an ethanol series. The dehydrated samples were critical point-dried (CPD 020, Balzers Union, Furstentum, Liechtenstein) and mounted onto aluminum stubs with double sided tape. The mounted samples were coated with gold by use of a 108 auto sputter coater (Cressington, Valencia, PA) and observed with a XL30 FEG microscope (Philips, Eindhoven, The Netherlands).
Immunofluorescence Microscopy-Pollen grains were fixed in a fixation solution (4% [w/v] formaldehyde, 0.1 M sodium phosphate buffer, pH 7.0) at room temperature for 1 h. They were washed with 0.1 M sodium phosphate buffer, pH 7.0, and 0.05% (v/v) Triton X-100 three times for 10 min each. They were blocked with 5% (w/v) nonfat dry milk in PBS for 2 h and allowed to react with rabbit antibodies against the maize pollen coat 35-kDa XYN1 (described earlier, in 1:100 dilutions) in PBS containing 1.67% (w/v) nonfat dry milk at 4°C overnight. After being washed with PBST (PBS containing 0.05% (v/v) Triton X-100) q3 times for 10 min each, the pollen grains were incubated with cyanine 5-conjugated donkey secondary antibodies against rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) at room temperature for 3 h. After being washed with PBST three times for 10 min each, the pollen grains were mounted in an antifade reagent from the SlowFade Antifade kit (Molecular Probes, Eugene, OR) and viewed under a Leica SP2 UV confocal microscope and photographed. Cyanine 5 was excited with 633-nm line, and its emission was detected at 650 -750 nm.

Transgenic Lines Containing No Xylanase on Pollen Were
Generated by an Antisense Technique-No T-DNA insertion mutant of XYN1 of maize or rice was available for study of the phenotype of xylanase-less pollen. Thus, we used an antisense technique to knock-out or knockdown the maize pollen coat xylanase. An antisense construct containing a 1.2-kb XYN1 promoter region, a 744-bp ORF encoding the removable, N-terminal 248 residues of the 60-kDa pre-xylanase, and a 931-bp antisense ORF encoding the mature 35-kDa xylanase (Fig. 1A) was transformed into maize immature embryos. We used the ORF encoding the mature 35-kDa xylanase instead of FIGURE 1. Antisense XYN1 construct, detection of its presence in transgenic calli and T 2 plants, and detection of XYN1, glucanase, and lipids in pollen. A, the antisense DNA construct, which consisted of a 1.2-kb XYN1 5Ј-nontranscribed sequence (promoter), a 0.7-kb sequence encoding the removable, N-terminal portion of pre-XYN1, a 0.9-kb XYN1 antisense sequence encoding the mature 35-kDa XYN1, and a nopaline synthase terminator (Nos-ter). Ts is the transcription initiation site, and N, B, E, and S are the NotI, BamHI, EcoRI, and SacI restriction sites, respectively. The 3.0-kb PCR fragment was that detected in transgenic calli described in B. The 0.5-kb DNA probe was used for DNA hybridization in C. B, PCR analyses of genomic DNA from calli induced from different embryos that had been co-bombarded with plasmid containing the antisense XYN1 construct and plasmid containing the bar gene, a selectable marker. Plasmid DNA containing the antisense XYN1 construct was used as a positive control (P). Genomic DNA extracted from calli bombarded only with a plasmid containing the bar gene was used as a negative control (N). A 3.0-kb fragment (marked with an arrow on the right margin) was amplified from the positive control and calli b, d, and h. Other samples (a, c, e, f, g, and i) contained no amplified fragment or fragments indicative of improperly inserted or modified antisense XYN1 construct. Molecular-length markers (M) were on the opposite margin lanes, and molecular length positions are shown on the left margin. C, hybridization of gDNA for the antisense XYN1 fragment and immunoblot detection of pollen XYN1 from the wild-type (wt) and various T 2 transgenic lines produced from selfing of the same T 1 line. The gDNA was digested with EcoRI and allowed to hybridize to a 0.5-kb probe (shown in A), which could hybridize the native XYN1 (indicated with an arrow on the right side) and the antisense fragment (all other visible fragments). Pollen from the same wild-type and T 2 transgenic lines were subjected to SDS-PAGE and immunoblot analyses with antibodies against XYN1 (details to be described in D). Only some of the T 2 transgenic lines lost the 35-kDa XYN1 completely or partially. D, SDS-PAGE of wild-type (wt) pollen coat and total extract of the wild type (left panel), SDS-PAGE, and immunoblot analyses of pollen total extract of the wild-type and a xyl-less line (middle panel), and TLC of lipids of the pollen coat of the wild-type and a xyl-less line (right panel). Left panel, SDS-PAGE showed the wild-type pollen coat (pc) possessing 70-kDa glucanase, 35-kDa XYN1, and a minor protein of 27 kDa; these proteins represented a very small fraction of the proteins in the total pollen extract (loading amounts were 20ϫ and 1ϫ an equal amount of pollen). Positions of molecular markers are shown on the left. Middle panel, SDS-PAGE and immunoblot analyses of total extract of wild-type and xyl-less pollen with anti-glucanase (GLA1) or anti-XYN1 revealed the selective absence of the 35-kDa XYN1 in xyl-less pollen. A pollen interior 55-kDa protein of unknown identity that reacted with the XYN1 antibody preparation (21) was observed and served as an indicator for equal loading of the wildtype and xyl-less pollen samples (also in C). Right panel, TLC of lipids extracted from wild-type and xyl-less pollen coat illustrated no major difference between the 2 samples. Lipid standards were C-29 alkane, cholesteroyl palmitate, and triolein (from top to bottom). the whole 60-kDa pre-xylanase to make the antisense construct because of the availability of the former DNA fragment, which we employed earlier to prepare the mature xylanase in bacteria for antibody preparation (14). We included the ORF encoding the sense N-terminal, removable 25-kDa fragment, whose function is unknown, to mimic the native 5Ј terminus of the mRNA. Several calli initiated from transformed embryos after selection culturing contained the antisense construct in the proper size and orientation (Fig. 1B); they were regenerated into adult plants (T 0 ). T 0 plants were pollinated with wild-type pollen to generate T 1 seeds. T 1 plants were checked for the contents of pollen coat xylanase, and those that had no detectable xylanase were retained. These T 1 plants were selfed to produce T 2 seeds. T 2 plants were found to have many copies of the antisense XYN1 per native XYN1. Although T 2 plants generated from one individual T 1 line possessed many copies of the antisense XYN1 in an identical pattern of restriction fragments, they produced pollen having highly variable XYN1 quantities. Exemplified results of T 2 plants generated from selfing of one T 1 plant are shown in Fig. 1C. Different T 2 plants possessed 10 -30 copies of the antisense XYN1 per native XYN1 in an identical pattern of restriction fragments (digestion with EcoRI) but highly variable amounts of pollen XYN1. These latter amounts ranged from 100% of that in the wild type to a non-detectable quantity.
The non-matching between the identical pattern of antisense XYN1 restriction fragments and the variable pollen XYN1 in the T 2 plants generated from selfing of one T 1 plant could be explained as follows. These T 2 plants might have obtained via Mendelian inheritance different pattern of the antisense fragments that was not detectable by the DNA hybridization procedure with EcoRI digestion; however, digestion with HindIII instead of EcoRI also revealed an identical pattern of the antisense fragments in them (data not shown). Alternatively, the T 2 plants might have undergone different epigenetic events (DNA and/or histone methylation), such that their antisense fragments were subjected to different degrees of silencing. This silencing could have occurred because the multiple copies of the antisense fragment generated from transcription formed double-stranded RNAs automatically or through RNAdependent RNA polymerase action, and the double-stranded RNAs were processed by Dicer into small interfering RNAs that could direct DNA methylation (27,28).
Because of the uncertainty of which of the T 2 lines generated by selfing of the same T 1 plant produced pollen with no pollencoat xylanase, we adopted the following strategy. We grew different T 2 transgenic lines to maturity, collected organ and pollen samples and preliminarily processed them for future experiments, then estimated the amounts of pollen coat xylanase in these lines. T 2 transgenic lines whose pollen contained no detectable xylanase (conservatively termed 0 -20% of the wild-type pollen xylanase) were used for further study. They are termed xyl-less lines, and their pollen, xyl-less pollen. We detected the amount of xylanase by immunoblotting rather than analyzing its enzyme activity because the amount of pollen coat xylanase from one T 2 plant was insufficient for accurate enzyme activity determination, germination test, and other examinations. Vegetative growth of xyl-less plants in greenhouses was indistinguishable from that of wild-type plants.
In three xyl-less lines studied, reverse transcriptase-PCR revealed the antisense transcript of the proper size and orientation in anthers. However, it was also present, albeit in smaller quantities, in leaves, immature kernels, and roots (data not shown). One possibility is that our 1.2-kb XYL1 promoter sequence, which contained a putative tapetumspecificity motif sequence (14), was not sufficient to confer high anther/tapetum specificity. In wild-type plants, the XYL1 transcript was present only in the tapetum among diverse tissues and organs (14). Thus, in xyl-less plants, the presence of the antisense construct in other organs did not affect their vegetative growth or our experimental purposes.

Xyl-less Lines and the Wild Type Exhibited No Appreciable Difference in Anther Structures by Electron Microscopy, but
Xyl-less Pollen Exhibited No Coat Xylanase by Immunofluorescence Microscopy-Transmission electron microscopy of anthers of various developmental stages, and mature pollen of xyl-less lines and the wild type revealed no difference in cell and subcellular morphology. As anthers matured, the tapetum cells gradually lost their cell wall, especially on the side facing the locule ( Fig. 2A). They acquired the characteristics of secretory cells by having abundant rough endoplasmic reticulum and secretory vesicles located toward the locule side. Microspores in anthers of xyl-less lines and the wild type underwent similar developmental changes ( Fig.  2A) as described in the first paragraph under "Experimental Procedures." Scanning EM also revealed no appreciable difference in shape and surface morphology, including the aperture, between xyl-less and wild-type pollen (Fig. 2B). The pollen aperture had a central exine that did not cover an annular area.
Nevertheless, immunofluorescence microscopy showed that xyl-less pollen had little or no surface xylanase, which was present on wild-type pollen (Fig. 2C), and the result reiterates our biochemical findings (Fig. 1D). Both xyl-less and wild-type pollen had the same amount of coat ␤-glucanase and the same amount and type of lipids (Fig. 1D).

Xyl-less Pollen Germinated in Vitro as Efficiently as Wildtype Pollen Did in a Liquid Medium but Less So on Gel Media
of Increasing Solidity-Germination in the current study is defined as observable protrusion of the tube and not tube elongation. A maize pollen grain does not change its shape upon water uptake to initiate germination, unlike the pollen grains of Brassicaceae species. Thus, we could not use swelling as a simple indicator of water uptake and germination but had to observe every pollen grain carefully for tube protrusion. Xyl-less pollen germinated as well as wild-type pollen did in liquid medium; ϳ80% of the pollen germinated within 20 min (Fig. 3, A and B). These percents of germination of wild-type pollen in liquid medium are similar to those reported earlier (8,29). Both types of pollen germinated less efficiently on gel media of increasing solidity, xyl-less pollen more so than wild-type pollen. On a 0.7% (w/v) gel, 17% of xyl-less and 33% of wild-type pollen germinated in 20 min. The disparity in percent germination of the two types of pollen became more pronounced, with 48% to 56% to 67% germination, when the solidity of the gel increased from 0.7% to 1.4% to 2.1%, respectively (Fig. 3, A and B). The percents of germination of both types of pollens on gel media increased with time, and the disparity gradually disappeared. A time course of pollen germination on 2.1% gel is exemplified in Fig. 3C.
Results shown in Fig. 3 and to be described that deal with pollen germination and tube penetration into silk were from experiments performed at least three times. Results of a similar pattern were observed, and one set of the results is shown in this report.
After Germination in Liquid or on Gel Media, Xyl-less Pollen Tubes Elongated as Rapidly as Wild-type Pollen Tubes Did-Xyl-less pollen germinated in vitro as efficiently as wild-type pollen did in a liquid medium but less so on gel media of increasing solidity (Fig. 3B). However, after germination, the tubes of both types of pollen elongated at a similar rate. Fig.  4 shows the elongation rates of the 2 types of pollen placed on 2.1% gel. Even though xyl-less pollen germinated slower than wild-type pollen did (Figs. 3C and 4), the tubes from both types of germinated pollen elongated at a similar rate. Thus, the disparity of the two types of pollen on gel media (Fig. 3B) applies only to initiation of germination (i.e. protrusion of the tube) rather than subsequent tube elongation.

Xyl-less Pollen Germinated on Silk Less Efficiently than Wild-type Pollen Did, and This Defect Could Not be Overcome by Application of Xylanase to the Silk-Xyl-less
pollen germinated on silk less efficiently than wild-type pollen did (Fig. 3B). We tested whether the addition of external xylanase would alleviate the low germination rate of xyl-less pollen. We could not add external xylanase directly to the surface of xyl-less pollen because the viability of maize pollen is severely affected by many factors (29). In our preliminary test, treatment of wild-type pollen with a blank solution briefly (e.g. 5 min) reduced its germination rate by more than 50%. Therefore, we soaked the silk for 5 min in a solution containing commercial Thermomyces xylanase, BSAs, or no extra ingredient (blank) and allowed the two types of pollen to germinate on treated and then briefly dried silk. Silk treated with a blank solution was a slightly less favorable medium for germination for both types of pollen (Fig. 5B). The addition of xylanase or BSA to the silk did not alleviate the low germination rate of xyl-less pollen (Fig. 5, A and B). This lack of alleviation suggests that for the coat xylanase to aid water uptake for germination, it has to be intimately associated with the pollen surface.  1 and 2) containing a conspicuous nucleus and packed cytoplasm adjacent to the locule (light region on the right) and (lower panels) a portion of a stage Ϫ5 microspore containing both intine (I) and exine (E). B, scanning EM images showing a mature pollen grain with an aperture and coarse surface (upper panels) and a magnified pollen surface with a granular structure (lower panels). C, fluorescence microscopy images showing pollen treated with anti-XYN1 IgG and then anti-IgG conjugated to cyanine 5 observed with a bright-field (upper panels) or fluorescence (lower panels) setting. Brightfield and fluorescence imaging of the wild-type and xyl-less pollen used the same confocal laser scanning microscopy settings (laser power and detection gain). In the fluorescence images, the background of the wildtype sample was faintly lighter than the xyl-less sample because of the fluorescence emitted from the pollen.

Xyl-less Pollen Tubes Penetrated Silk Less Efficiently than
Wild-type Pollen Tubes Did, and This Defect Could Be Overcome by Application of Xylanase to the Silk-When xyl-less and wild-type pollen were placed on silk, they germinated and their tubes elongated. After 20 min, the tubes began to penetrate the silk into the carpel interior. Xyl-less pollen tubes did not penetrate the silk as efficiently as wild-type pollen tubes did (Fig. 6, A  and B). Xyl-less pollen tubes that failed to penetrate the silk elongated along (Fig. 6A) or curved at the silk surface. After 60 min, ϳ40% of the xyl-less pollen tubes, compared with ϳ70% of the wild-type pollen tubes, penetrated the silk. These percents reached ϳ50 and ϳ90%, respectively, after 90 min. Prior application of xylanase to the silk restored the ability of xyl-less pollen tubes to penetrate to almost the same level as the wild-type pollen tubes. The restoration was ineffective when BSA or a blank solution instead of xylanase was pre-applied to the silk. Thus, loss of xylanase on xyl-less pollen was the cause of the reduced ability of the tubes to penetrate the silk.

The Efficiency of Germination on and Tube Penetration into Silk of Pollen from Different Transgenic Lines and the Wild Type
Were Dependent on the Amount of Coat Xylanase-In the current study, with the exception of this section, we examined germination on and tube penetration into silk of pollen of transgenic lines with 0 -20% xylanase (termed xyl-less pollen). We repeated our examinations by comparing pollen of the wild type (100%) and transgenic lines containing 40 -60% (termed 50% for convenience) and 0 -20% (termed 10%, only in this section) coat xylanase. Fig. 7 shows that pollen from the two transgenic lines exhibited reduced efficiency in germination on and tube penetration into silk after 90 min, depending on the residual amounts of xylanase.
For Wild-type Pollen, Xylose Inhibited the Coat Xylanase Activity in Vitro but Did Not Affect Germination in a Liquid Medium, and Application of Xylose to Silk Hindered Pollen Tube Penetration-Ideally, we would test coat xylanase enzymatic activity of wild-type pollen on silk. Technical difficulties complicated such a direct detection. The xylanase activity is an endohydrolase, and free xylose would not be released and detected. Observing pollen action on silk at a physiological condition would necessitate application of a limited number of pollen grains per silk area, and the expected minimal number of xylose ends generated from xylan breakage in the silk wall would hinder biochemical detection. Pollen coat also contains a FIGURE 6. Penetration of tubes of germinated wild-type (wt) and xyl-less pollen into silk. A, light microscopy of pollen germinated on silk for 90 min observed with use of a bright-field or fluorescence setting after the samples had been treated with aniline blue. One wild-type pollen tube that penetrated and one xyl-less pollen tube that did not are shown. B, percentage of tubes of germinated pollen that penetrated into silk untreated or treated with xylanase, BSA, or a blank solution at time intervals. The S.E. values were calculated as described under "Experimental Procedures." Pollen of wild-type and transgenic lines with 40 -60% (labeled as 50%; e.g. pollen from T 2 line 75b shown in Fig. 1C) and 0 -20% (labeled as 10% in this figure but termed xyl-less in other parts of this report; e.g. pollen from T 2 line 80a shown in Fig. 1C) wild-type xylanase were used. Percentage of germination of pollen on silk (left portion) and penetration of tubes from germinated pollen into silk (right portion) were recorded after 90 min. S.E. values were calculated as described under "Experimental Procedures." The difference in germination (and tube penetration) between any two of the three pollen samples was significant (p Ͻ 0.05).
glucanase, which could hydrolyze glucan of cellulose and complicate the detection of breakage of xylan in the hemicellulose. To circumvent these difficulties, we took advantage of our observation that the pollen coat xylanase enzymatic activity was inhibited by xylose but not glucose and tested the effect of xylose on pollen germination and tube penetration into silk.
Xylose inhibited pollen coat xylanase enzyme activity on xylan in vitro (Fig. 8A). In this enzyme activity assay, we wanted to add xylose to the assay medium and therefore could not use the common assay procedure of detecting the released xylose or the exposed reducing ends on xylan by colorimetry. Instead, we used a viscosity assay procedure, which detected the reduction in viscosity of a xylan resuspension after it had been broken down into smaller chains by endoxylanase activity (14,30). In our assay, xylanase activity on oat spelled xylan was detected in the presence of coat xylanase, or Thermomyces xylanase, but not in the absence of enzyme. The coat xylanase activity was inhibited by 0.1 M xylose but not glucose.
Although xylose inhibited coat xylanase enzymatic activity, it had no effect on germination of wild-type pollen in a liquid medium. Fig. 8B shows that wild-type pollen germinated similarly in a liquid medium in the absence or presence of 0.1 M xylose or glucose.
Also, addition of 0.1 M xylose or glucose to silk did not affect pollen germination on the treated silk (Fig. 8C). As noted earlier (Fig. 5B), pretreatment of silk with a blank solution slightly reduced the percentage of pollen germination on the silk after 90 min. Regardless, pretreatment of silk with xylose, glucose, and a blank solution all slightly reduced the percentage of pollen germination to a similar extent.
However, pretreatment of silk with 0.1 M xylose but not glucose inhibited tube penetration into the silk (Fig. 8C). The findings show that xylanase on maize pollen aids penetration of the tube into silk because of its xylanase enzyme activity on the silk wall.

Three Different Molecular Actions of Pollen Coat Ingredients on the Stigma Have Been
Recognized-Recent studies have recognized three molecular actions of pollen on the stigma. First, initiation of signal transduction for water uptake in self-incompatibility systems has been delineated (5-6). Second, the physical requirements of pollen-surface materials for water uptake/ germination have been documented. A mutational loss of a pollen coat lipid (a common phenotype in many Arabidopsis wax mutants described by the Arabidopsis Biological Resource Center) or protein including oleosin (31) and xylanase (this report) leads to a reduced ability of the mutant pollen to take up water and thus undergo fertilization. Third, as documented in the current report, the enzymatic action of xylanase on the stigma xylan creates an opening for tube penetration. Numerous cell wall digestive enzymes and modulating proteins are known to be on the pollen surface or quickly released from hydrated pollen, but their action on the stigma wall has not been previously reported. Some of these proteins appear to exert their action not on the stigma but in the pollen tube track in the style (18).
In the current experiments, tubes of xyl-less pollen were still able to penetrate silk into the carpel interior, even though the penetration efficiency had been substantially reduced (by ϳ50% in xyl-less versus wild-type pollen; Fig. 6). Two possible explanations are offered. First, the xyl-less pollen we used contained 0 -20% of the wild-type pollen xylanase, even though we observed no xylanase on the immunoblot. A trace amount of leftover xylanase would still be able to perform wall hydrolysis, albeit with less efficiency. Second, the tapetum-synthesized pollen coat materials in maize include two other, even though less abundant, hydrolases (a ␤-glucanase and a protease) ( Fig.  1D; Ref. 17). Whether these other hydrolases are leftover tapetum proteins with no further function or are required to act collaboratively or synergistically with the xylanase for optimal hydrolysis of the silk wall remains to be elucidated.
Loss of Pollen Coat Xylanase Impairs Water Uptake on Silk or Solid Media-Xyl-less pollen germinated as efficiently as wildtype pollen did in a liquid medium but less so on gel media of increasing solidity (Fig. 3). This finding suggests that the presence of xylanase on the pollen surface provides efficient water uptake into pollen for germination. After germination in a liquid or on a gel medium (Fig. 4), xyl-less pollen tubes elongated as rapidly as wild-type pollen tubes did. Presumably, germination involves uptake of water through the aperture of a pollen grain; xylanase and other hydrophilic components on the pollen surface help with contact of the grain to the medium and movement of water along the pollen surface to the aperture. After germination, water is taken up directly and easily into the pollen tube and not through the aperture, and thus the presence of xylanase on the pollen becomes irrelevant.
The xylanase molecule is overall very hydrophilic, as revealed in a hydropathic plot (14). Our calculation shows that a pollen grain of 70-m diameter has 5.8 pg of the 35-kDa xylanase. If we assume that a pollen grain is completely spherical and has xylanase evenly distributed on its surface and that the xylanase molecule has a globular structure, the xylanase molecules would occupy about 10% of the pollen surface. Therefore, the coat xylanase aiding direct water uptake should be minimal. The pollen surface also contains flavonoids (ϳ400 pg per pollen; Ref. 32) and perhaps sugars and amino acids in greater abundance. These other hydrophilic or amphipathic molecules on pollen should be the major components mediating direct water uptake.
Xyl-less pollen germinated less efficiently than wild-type pollen did on silk and gel media of increasing solidity. This loss of efficiency should have more to do with the contact of the pollen grain surface with the solid media than to a slight decrease in the osmotic potential of the gel of increasing solidity (the osmotic potentials of the media containing 0, 0.7, 1.4, and 2.1% gel were Ϫ1.34, Ϫ1.56, Ϫ1.67, and Ϫ1.78 MPa, respectively, determined with a Wescor Dew Point Microvoltmeter), because pollen has a very low water potential (Ϫ20 MPa in maize pollen; Ref. 33). The pollen grain has a granular, coarse surface (Fig. 2B), and thus water uptake depends greatly on its proper contact with the solid medium. It is unlikely that the loss of xylanase from the exine surface affected the membrane or water potential of the pollen. Both wild-type and xyl-less pollen grains rapidly burst at similar rates when placed in water, with their protoplasts disintegrating after emergence from the aperture (data not shown). The reduced water uptake efficiency of xyl-less pollen is similar to that of mutant pollen of other species with a lost coat ingredient, such as lipid (many wax mutants, described by the Arabidopsis Biological Resource Center) or protein (31). We suggest that the loss of xylanase, or another protein or lipid, on the pollen surface would impair the semifluid and amphipathic status of the coat materials, optimized by evolution or breeding, to act as a waterproofing agent and also a wick for water uptake. The main function of xylanase is related to its enzymatic activity on tube penetration into silk.
Wall Hydrolases and Modulating Proteins on the Pollen Surface Crest Can Be Divided into Two Functional Groups with Distinct Origins-Maize pollen is the best studied system in terms of wall hydrolases and modulating proteins associated with the pollen surface crest. Findings presented in the current and earlier reports have allowed us to divide these proteins into two groups. Members of one group, including mostly the endoxylanase, are synthesized in the tapetum cells and transferred to the microspore surface as coat materials (14,17). Members of the other group, including polygalacturonase and wall-modulating proteins such as extensin and calcium-chelating proteins, are synthesized in and secreted from the pollen interior during late maturation of pollen (microspore) or after the mature pollen have germinated (17). We suggest that the tapetum-synthesized endoxylanase on pollen is responsible for the initial hydrolysis of the carpel wall xylan after pollen landing, thus creating an opening for entry of the pollen tube, and that the pollen-synthesized hydrolases and wall-modulating proteins are for the subsequent hydrolysis and modulation of the carpel interior wall, thereby generating a path for tube advancement to the ovary. The pollen-synthesized hydrolases and wall-modulating proteins could also function to loosen the pollen tube wall for growth, generate sugars as wall precursors for tube growth, and produce oligosaccharides as signal molecules. These potential functions of the pollen-synthesized polygalacturonase (34) and pectin methylesterase (35) in the pollen tube track have been suggested or described.