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

Abstract 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.