The Predominant Protein on the Surface of Maize Pollen Is an Endoxylanase Synthesized by a Tapetum mRNA with a Long 5′ Leader (1999)

In plants, the pollen coat covers the exine wall of the pollen and is the outermost layer that makes the initial contact with the stigma surface during sexual reproduction. Little is known about the constituents of the pollen coat, especially in wind-pollinated species. The pollen coat was extracted with diethyl ether from the pollen of maize (Zea mays L.), and a predominant protein of 35 kDa was identified. On the basis of the N-terminal sequence of this protein, a cDNA clone of theXyl gene was obtained by reverse transcriptase-polymerase chain reaction. The deduced amino acid sequence of the 35-kDa protein shared similarities with the sequences of many microbial xylanases and a barley aleurone-layer xylanase. The 35-kDa protein in the pollen-coat extract was purified to homogeneity by fast protein liquid chromatography and determined to be an acidic endoxylanase that was most active on oat spelt xylan. Northern and in situhybridization showed that Xyl was specifically expressed in the tapetum of the anther after the tetrad microspores had become individual microspores. Southern hybridization and gene-copy reconstruction studies showed only one copy of the Xyl gene per haploid genome. Analyses of the genomic DNA sequence ofXyl and RNase protection studies with the transcript revealed many regulatory motifs at the promoter region and an intron at the 5′ leader region of the transcript. The Xyl transcript had a 562-nucleotide (nt) 5′ leader, a 54-nt sequence encoding a putative signal peptide, a 933-nt coding sequence, and a 420-nt 3′-untranslated sequence. The unusually long 5′ leader had an open reading frame encoding a putative 175-residue protein whose sequence was most similar to that of a microbial arabinosidase. The maize xylanase is the first enzyme documented to be present in the pollen coat. Its possible role in the hydrolysis of the maize type II primary cell wall (having xylose, glucose, and arabinose as the major moieties) of the tapetum cells and the stigma surface is discussed. In plants, the pollen coat covers the exine wall of the pollen and is the outermost layer that makes the initial contact with the stigma surface during sexual reproduction. Little is known about the constituents of the pollen coat, especially in wind-pollinated species. The pollen coat was extracted with diethyl ether from the pollen of maize (Zea mays L.), and a predominant protein of 35 kDa was identified. On the basis of the N-terminal sequence of this protein, a cDNA clone of theXyl gene was obtained by reverse transcriptase-polymerase chain reaction. The deduced amino acid sequence of the 35-kDa protein shared similarities with the sequences of many microbial xylanases and a barley aleurone-layer xylanase. The 35-kDa protein in the pollen-coat extract was purified to homogeneity by fast protein liquid chromatography and determined to be an acidic endoxylanase that was most active on oat spelt xylan. Northern and in situhybridization showed that Xyl was specifically expressed in the tapetum of the anther after the tetrad microspores had become individual microspores. Southern hybridization and gene-copy reconstruction studies showed only one copy of the Xyl gene per haploid genome. Analyses of the genomic DNA sequence ofXyl and RNase protection studies with the transcript revealed many regulatory motifs at the promoter region and an intron at the 5′ leader region of the transcript. The Xyl transcript had a 562-nucleotide (nt) 5′ leader, a 54-nt sequence encoding a putative signal peptide, a 933-nt coding sequence, and a 420-nt 3′-untranslated sequence. The unusually long 5′ leader had an open reading frame encoding a putative 175-residue protein whose sequence was most similar to that of a microbial arabinosidase. The maize xylanase is the first enzyme documented to be present in the pollen coat. Its possible role in the hydrolysis of the maize type II primary cell wall (having xylose, glucose, and arabinose as the major moieties) of the tapetum cells and the stigma surface is discussed. In plants, pollen is the male gametophyte that interacts with the female reproductive organ during sexual reproduction (1Bedinger P. Plant Cell. 1992; 4: 879-887PubMed Google Scholar, 2Mascarenhas J.P. Plant Cell. 1993; 10: 1303-1314Google Scholar, 3McCormick S. Plant Cell. 1993; 5: 1265-1275Crossref PubMed Scopus (459) Google Scholar, 4Goldberg R.B. Beals T.P. Sanders P.M. Plant Cell. 1993; 5: 1217-1229Crossref PubMed Scopus (603) Google Scholar). It is produced in the anther of a flower, where diploid pollen mother cells undergo meiosis to produce haploid cells, termed microspores. Microspores mature in the anther locule, which is an enclosed, extracellular fluid space, to become pollen. During their maturation (microsporogenesis), the microspores interact with the tapetum, which consists of one layer of metabolically active cells enclosing the locule. The tapetum serves several important roles in the maturation of the microspores (5Pacini E. Franchi G.G. Plant Syst. Evol. (suppl.). 1993; 7: 1-11Crossref Google Scholar). Throughout microsporogenesis, the tapetum transfers nutrients to, and regulates the composition of, the locule fluid. In addition, at an early stage of microspore maturation, the tapetum secretes β-glucanase, which hydrolyzes the β-glucan (callose) cell wall enclosing the microspore tetrads (6Frankel R. Izhar S. Nitsan J. Biochem. Genet. 1969; 3: 451-455Crossref PubMed Scopus (65) Google Scholar). The released individual microspores continue to mature in the locule. Midway through microsporogenesis, the tapetum secretes exine precursors, which are condensed on the surface of the pollen inner wall (the intine) to form the outer wall (the exine). The exine has many bacules arranged into species-specific patterns, and the bacules generate cavities on the pollen surface. At a late stage of microsporogenesis, the tapetum produces other molecules, which will be deposited in the exine cavities and on the exine surface. These pollen surface molecules are called pollen coat, tryphine, or pollenkitt. After pollination, the pollen lands on the stigma of the carpel and produces a pollen tube, which penetrates into the stigma and eventually delivers the male gametes to the ovules.The pollen coat is the outermost layer of the pollen and will make the initial contact with the stigma surface during sexual reproduction. It may serve various functions, such as protecting the pollen from solar radiation, giving the pollen color, preventing water loss from pollen, attracting insect pollinators, adhering the pollen to each other and to the pollinating insect bodies and the stigma surface, aiding hydration and thus the initial germination of the pollen, and exerting self-compatibility and incompatibility (5Pacini E. Franchi G.G. Plant Syst. Evol. (suppl.). 1993; 7: 1-11Crossref Google Scholar). In view of these possible species-specific functions, the production of the pollen-coat materials in the tapetum may be a highly regulated biosynthetic process rather than a passive process involving remnants of dead tapetum cells.Currently, information on the constituents of the pollen-coat materials is limited. Slightly more information is available on constituents of the exine and the intine underlining the pollen coat. The exine has sporopollenin as its structural constituents, and it contains many different Plant 1992; PubMed Scopus Google Scholar). The intine contains several J. Scopus Google Plant PubMed Scopus Google J. S. Plant PubMed Scopus Google and P. R. Plant PubMed Scopus Google and other R.B. J. J. Scholar). intine that more are by in the pollen, and the are released from the to the The known pollen are present in the of the pollen and on the pollen surface R.B. J. PubMed Scopus Google Scholar). the constituents of the exine and intine of the pollen wall in the pollen coat that are deposited on the surface of these pollen are the surface and of the pollen coat by several J. S. 1993; Scopus Google Scholar, Plant Cell. PubMed Scopus Google Scholar, S. PubMed Scopus Google Scholar, Plant J. PubMed Scopus Google Scholar). The pollen coat be extracted with an such as or diethyl and the the pollen which be with the The pollen coat contains as the major constituents, which in the of the tapetum cells at the stage of anther S. PubMed Scopus Google Scholar). The predominant in the pollen coat are to which as structural on the bodies of Plant 1992; Scopus Google Scholar). In the in of the tapetum cells S. PubMed Scopus Google Scholar). the are specifically and their are released from the and the tapetum cells to the pollen surface. The of the and in the pollen coat are similar to on obtained from to a J. Plant J. 1993; 3: PubMed Scopus Google Scholar). of in long chain produces pollen pollen these pollen on the stigma 1993; 7: PubMed Scopus Google Scholar). and are or and their pollen has a coat, which is and contains In pollen of wind-pollinated such as the has of pollen coat, which are and a of The pollen coat in the wind-pollinated be different from that in or in constituents and the pollen coat from a wind-pollinated species. pollen coat contains a of and one protein protein is an endoxylanase by a copy gene expressed in the The gene transcript has an unusually long 5′ leader, a long open reading frame open reading fast protein liquid liquid reverse transcriptase-polymerase chain open reading fast protein liquid liquid reverse transcriptase-polymerase chain which an arabinosidase. has a II cell which contains and with xylose, and Plant J. 1993; 3: PubMed Scopus Google Scholar). The of a xylanase and a putative may important in the hydrolysis of the primary cell wall in of sexual such as the initial of the pollen into the stigma maize pollen coat xylanase is in the it has a signal and is to the locule the the tapetum β-glucanase, which is to the locule the hydrolysis of the cell wall of the microspore tetrad (6Frankel R. Izhar S. Nitsan J. Biochem. Genet. 1969; 3: 451-455Crossref PubMed Scopus (65) Google the xylanase is after the microspore tetrad has become microspores. It on the pollen surface, and and is the only enzyme documented to be present in the pollen coat, several other to be present in the intine of pollen These by are of in the pollen the maize xylanase is the of in the In addition, the maize xylanase is present and in the pollen coat, as revealed by of the maize pollen xylanase be the enzyme is in the hydrolysis of the wall of the microspore The cell of the tapetum cells are (5Pacini E. Franchi G.G. Plant Syst. Evol. (suppl.). 1993; 7: 1-11Crossref Google or during the of the xylanase the enzyme is in the hydrolysis of the tapetum cell or the cell wall anther it is that the enzyme is and and in the pollen coat. the xylanase may be deposited in the pollen coat to a role after the pollen has on the On the stigma surface, the enzyme may the cell wall and thus the initial of the pollen into the stigma The the of a the stigma a and J. Google Scholar). It is possible that the xylanase may regulatory from the stigma cells a to be regulatory one or more of the possible functions, the xylanase has the the maize cell wall has a II as a major Plant J. 1993; 3: PubMed Scopus Google and it is more active on the of oat than the xylanase may a similar to the which are and on the pollen surface, may as a water from the stigma pollen germination and initial In addition, a xylanase produced by as an in in the exerting Plant 1993; PubMed Scopus Google is the predominant protein in the pollen coat and is from the tapetum and in The pollen coat are the predominant and are specifically from the with in the tapetum late in Plant Cell. PubMed Scopus Google Scholar, S. PubMed Scopus Google Scholar, Plant J. PubMed Scopus Google Scholar). These on and make it that the pollen coat is produced from of dead tapetum cells during late the of the pollen coat and in the tapetum and their the microspores to be highly are only in known to be in the tapetum and into the locule microspore of these at a different during and has a The in is in the tapetum during early and to the locule, where it hydrolyzes the wall of the microspore the of the after the hydrolysis is (6Frankel R. Izhar S. Nitsan J. Biochem. Genet. 1969; 3: 451-455Crossref PubMed Scopus (65) Google Scholar). in maize is in the tapetum at a stage of and and deposited on the pollen it may the cell wall of the in are at a late stage of in the tapetum and after the microspore the of the in the pollen coat is S. PubMed Scopus Google Scholar, Plant J. PubMed Scopus Google only other xylanase gene is in barley P. Genet. PubMed Scopus Google Scholar). The barley gene is expressed in the layer during The enzyme is to the where it hydrolyzes the cell thus the to other from the of the barley xylanase gene are present in on a from In on genomic DNA and genomic clone and one copy which is specifically expressed in the the maize and barley xylanases an signal and are to the The 5′ leader of the xylanase in maize is unusually and that in barley has a the maize and barley sequences similarities the 5′ leader of the maize transcript and the 5′ of the barley It will be to the xylanases of the tapetum and the layer are by different and the transcript in the layer contains a long 5′ of a long 5′ leader sequence an in Xyl is It is that this an that is to the xylanase by the primary the 5′ leader produce an it will be to the in the cell wall in the sexual reproductive and their during it will be to the which is of a signal peptide, is from the tapetum cells to the locule. The is at the 5′ of the and the are by the produced as a the is into the a similar to that to be In plants, pollen is the male gametophyte that interacts with the female reproductive organ during sexual reproduction (1Bedinger P. Plant Cell. 1992; 4: 879-887PubMed Google Scholar, 2Mascarenhas J.P. Plant Cell. 1993; 10: 1303-1314Google Scholar, 3McCormick S. Plant Cell. 1993; 5: 1265-1275Crossref PubMed Scopus (459) Google Scholar, 4Goldberg R.B. Beals T.P. Sanders P.M. Plant Cell. 1993; 5: 1217-1229Crossref PubMed Scopus (603) Google Scholar). It is produced in the anther of a flower, where diploid pollen mother cells undergo meiosis to produce haploid cells, termed microspores. Microspores mature in the anther locule, which is an enclosed, extracellular fluid space, to become pollen. During their maturation (microsporogenesis), the microspores interact with the tapetum, which consists of one layer of metabolically active cells enclosing the locule. The tapetum serves several important roles in the maturation of the microspores (5Pacini E. Franchi G.G. Plant Syst. Evol. (suppl.). 1993; 7: 1-11Crossref Google Scholar). Throughout microsporogenesis, the tapetum transfers nutrients to, and regulates the composition of, the locule fluid. In addition, at an early stage of microspore maturation, the tapetum secretes β-glucanase, which hydrolyzes the β-glucan (callose) cell wall enclosing the microspore tetrads (6Frankel R. Izhar S. Nitsan J. Biochem. Genet. 1969; 3: 451-455Crossref PubMed Scopus (65) Google Scholar). The released individual microspores continue to mature in the locule. Midway through microsporogenesis, the tapetum secretes exine precursors, which are condensed on the surface of the pollen inner wall (the intine) to form the outer wall (the exine). The exine has many bacules arranged into species-specific patterns, and the bacules generate cavities on the pollen surface. At a late stage of microsporogenesis, the tapetum produces other molecules, which will be deposited in the exine cavities and on the exine surface. These pollen surface molecules are called pollen coat, tryphine, or pollenkitt. After pollination, the pollen lands on the stigma of the carpel and produces a pollen tube, which penetrates into the stigma and eventually delivers the male gametes to the The pollen coat is the outermost layer of the pollen and will make the initial contact with the stigma surface during sexual reproduction. It may serve various functions, such as protecting the pollen from solar radiation, giving the pollen color, preventing water loss from pollen, attracting insect pollinators, adhering the pollen to each other and to the pollinating insect bodies and the stigma surface, aiding hydration and thus the initial germination of the pollen, and exerting self-compatibility and incompatibility (5Pacini E. Franchi G.G. Plant Syst. Evol. (suppl.). 1993; 7: 1-11Crossref Google Scholar). In view of these possible species-specific functions, the production of the pollen-coat materials in the tapetum may be a highly regulated biosynthetic process rather than a passive process involving remnants of dead tapetum information on the constituents of the pollen-coat materials is limited. Slightly more information is available on constituents of the exine and the intine underlining the pollen coat. The exine has sporopollenin as its structural constituents, and it contains many different Plant 1992; PubMed Scopus Google Scholar). The intine contains several J. Scopus Google Plant PubMed Scopus Google J. S. Plant PubMed Scopus Google and P. R. Plant PubMed Scopus Google and other R.B. J. J. Scholar). intine that more are by in the pollen, and the are released from the to the The known pollen are present in the of the pollen and on the pollen surface R.B. J. PubMed Scopus Google Scholar). the constituents of the exine and intine of the pollen wall in the pollen coat that are deposited on the surface of these pollen are the surface and of the pollen coat by several J. S. 1993; Scopus Google Scholar, Plant Cell. PubMed Scopus Google Scholar, S. PubMed Scopus Google Scholar, Plant J. PubMed Scopus Google Scholar). The pollen coat be extracted with an such as or diethyl and the the pollen which be with the The pollen coat contains as the major constituents, which in the of the tapetum cells at the stage of anther S. PubMed Scopus Google Scholar). The predominant in the pollen coat are to which as structural on the bodies of Plant 1992; Scopus Google Scholar). In the in of the tapetum cells S. PubMed Scopus Google Scholar). the are specifically and their are released from the and the tapetum cells to the pollen surface. The of the and in the pollen coat are similar to on obtained from to a J. Plant J. 1993; 3: PubMed Scopus Google Scholar). of in long chain produces pollen pollen these pollen on the stigma 1993; 7: PubMed Scopus Google Scholar). and are or and their pollen has a coat, which is and contains In pollen of wind-pollinated such as the has of pollen coat, which are and a of The pollen coat in the wind-pollinated be different from that in or in constituents and the pollen coat from a wind-pollinated species. pollen coat contains a of and one protein protein is an endoxylanase by a copy gene expressed in the The gene transcript has an unusually long 5′ leader, a long open reading frame open reading fast protein liquid liquid reverse transcriptase-polymerase chain open reading fast protein liquid liquid reverse transcriptase-polymerase chain which an arabinosidase. has a II cell which contains and with xylose, and Plant J. 1993; 3: PubMed Scopus Google Scholar). The of a xylanase and a putative may important in the hydrolysis of the primary cell wall in of sexual such as the initial of the pollen into the stigma maize pollen coat xylanase is in the it has a signal and is to the locule the the tapetum β-glucanase, which is to the locule the hydrolysis of the cell wall of the microspore tetrad (6Frankel R. Izhar S. Nitsan J. Biochem. Genet. 1969; 3: 451-455Crossref PubMed Scopus (65) Google the xylanase is after the microspore tetrad has become microspores. It on the pollen surface, and and is the only enzyme documented to be present in the pollen coat, several other to be present in the intine of pollen These by are of in the pollen the maize xylanase is the of in the In addition, the maize xylanase is present and in the pollen coat, as revealed by of the maize pollen xylanase be the enzyme is in the hydrolysis of the wall of the microspore The cell of the tapetum cells are (5Pacini E. Franchi G.G. Plant Syst. Evol. (suppl.). 1993; 7: 1-11Crossref Google or during the of the xylanase the enzyme is in the hydrolysis of the tapetum cell or the cell wall anther it is that the enzyme is and and in the pollen coat. the xylanase may be deposited in the pollen coat to a role after the pollen has on the On the stigma surface, the enzyme may the cell wall and thus the initial of the pollen into the stigma The the of a the stigma a and J. Google Scholar). It is possible that the xylanase may regulatory from the stigma cells a to be regulatory one or more of the possible functions, the xylanase has the the maize cell wall has a II as a major Plant J. 1993; 3: PubMed Scopus Google and it is more active on the of oat than the xylanase may a similar to the which are and on the pollen surface, may as a water from the stigma pollen germination and initial In addition, a xylanase produced by as an in in the exerting Plant 1993; PubMed Scopus Google is the predominant protein in the pollen coat and is from the tapetum and in The pollen coat are the predominant and are specifically from the with in the tapetum late in Plant Cell. PubMed Scopus Google Scholar, S. PubMed Scopus Google Scholar, Plant J. PubMed Scopus Google Scholar). These on and make it that the pollen coat is produced from of dead tapetum cells during late the of the pollen coat and in the tapetum and their the microspores to be highly are only in known to be in the tapetum and into the locule microspore of these at a different during and has a The in is in the tapetum during early and to the locule, where it hydrolyzes the wall of the microspore the of the after the hydrolysis is (6Frankel R. Izhar S. Nitsan J. Biochem. Genet. 1969; 3: 451-455Crossref PubMed Scopus (65) Google Scholar). in maize is in the tapetum at a stage of and and deposited on the pollen it may the cell wall of the in are at a late stage of in the tapetum and after the microspore the of the in the pollen coat is S. PubMed Scopus Google Scholar, Plant J. PubMed Scopus Google only other xylanase gene is in barley P. Genet. PubMed Scopus Google Scholar). The barley gene is expressed in the layer during The enzyme is to the where it hydrolyzes the cell thus the to other from the of the barley xylanase gene are present in on a from In on genomic DNA and genomic clone and one copy which is specifically expressed in the the maize and barley xylanases an signal and are to the The 5′ leader of the xylanase in maize is unusually and that in barley has a the maize and barley sequences similarities the 5′ leader of the maize transcript and the 5′ of the barley It will be to the xylanases of the tapetum and the layer are by different and the transcript in the layer contains a long 5′ of a long 5′ leader sequence an in Xyl is It is that this an that is to the xylanase by the primary the 5′ leader produce an it will be to the in the cell wall in the sexual reproductive and their during it will be to the which is of a signal peptide, is from the tapetum cells to the locule. The is at the 5′ of the and the are by the produced as a the is into the a similar to that to be The maize pollen coat xylanase is in the it has a signal and is to the locule the the tapetum β-glucanase, which is to the locule the hydrolysis of the cell wall of the microspore tetrad (6Frankel R. Izhar S. Nitsan J. Biochem. Genet. 1969; 3: 451-455Crossref PubMed Scopus (65) Google the xylanase is after the microspore tetrad has become microspores. It on the pollen surface, and and is the only enzyme documented to be present in the pollen coat, several other to be present in the intine of pollen These by are of in the pollen the maize xylanase is the of in the In addition, the maize xylanase is present and in the pollen coat, as revealed by The of the maize pollen xylanase be the enzyme is in the hydrolysis of the wall of the microspore The cell of the tapetum cells are (5Pacini E. Franchi G.G. Plant Syst. Evol. (suppl.). 1993; 7: 1-11Crossref Google or during the of the xylanase the enzyme is in the hydrolysis of the tapetum cell or the cell wall anther it is that the enzyme is and and in the pollen coat. the xylanase may be deposited in the pollen coat to a role after the pollen has on the On the stigma surface, the enzyme may the cell wall and thus the initial of the pollen into the stigma The the of a the stigma a and J. Google Scholar). It is possible that the xylanase may regulatory from the stigma cells a to be regulatory one or more of the possible functions, the xylanase has the the maize cell wall has a II as a major Plant J. 1993; 3: PubMed Scopus Google and it is more active on the of oat than the xylanase may a similar to the which are and on the pollen surface, may as a water from the stigma pollen germination and initial In addition, a xylanase produced by as an in in the exerting Plant 1993; PubMed Scopus Google Scholar). is the predominant protein in the pollen coat and is from the tapetum and in The pollen coat are the predominant and are specifically from the with in the tapetum late in Plant Cell. PubMed Scopus Google Scholar, S. PubMed Scopus Google Scholar, Plant J. PubMed Scopus Google Scholar). These on and make it that the pollen coat is produced from of dead tapetum cells during late the of the pollen coat and in the tapetum and their the microspores to be highly are only in known to be in the tapetum and into the locule microspore of these at a different during and has a The in is in the tapetum during early and to the locule, where it hydrolyzes the wall of the microspore the of the after the hydrolysis is (6Frankel R. Izhar S. Nitsan J. Biochem. Genet. 1969; 3: 451-455Crossref PubMed Scopus (65) Google Scholar). in maize is in the tapetum at a stage of and and deposited on the pollen it may the cell wall of the in are at a late stage of in the tapetum and after the microspore the of the in the pollen coat is S. PubMed Scopus Google Scholar, Plant J. PubMed Scopus Google Scholar). The only other xylanase gene is in barley P. Genet. PubMed Scopus Google Scholar). The barley gene is expressed in the layer during The enzyme is to the where it hydrolyzes the cell thus the to other from the of the barley xylanase gene are present in on a from In on genomic DNA and genomic clone and one copy which is specifically expressed in the the maize and barley xylanases an signal and are to the The 5′ leader of the xylanase in maize is unusually and that in barley has a the maize and barley sequences similarities the 5′ leader of the maize transcript and the 5′ of the barley It will be to the xylanases of the tapetum and the layer are by different and the transcript in the layer contains a long 5′ The of a long 5′ leader sequence an in Xyl is It is that this an that is to the xylanase by the primary the 5′ leader produce an it will be to the in the cell wall in the sexual reproductive and their during it will be to the which is of a signal peptide, is from the tapetum cells to the locule. The is at the 5′ of the and the are by the produced as a the is into the a similar to that to be and in the of the and