Bih F Y, Wu S S, Ratnayake C, Walling L L, Nothnagel E A, Huang A H
Department of Botany and Plant Sciences, University of California, Riverside, California 92521, USA.
J Biol Chem. 1999 Aug 6;274(32):22884-94. doi: 10.1074/jbc.274.32.22884.
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 the Xyl 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 situ hybridization 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 of Xyl 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.
在植物中，花粉壁覆盖在花粉的外壁上，是有性生殖过程中与柱头表面进行初次接触的最外层结构。关于花粉壁的成分，人们了解甚少，尤其是在风媒传粉的物种中。用乙醚从玉米（Zea mays L.）花粉中提取花粉壁，鉴定出一种主要的35 kDa蛋白质。根据该蛋白质的N端序列，通过逆转录聚合酶链反应获得了Xyl基因的cDNA克隆。推导的35 kDa蛋白质的氨基酸序列与许多微生物木聚糖酶和大麦糊粉层木聚糖酶的序列具有相似性。通过快速蛋白质液相色谱法将花粉壁提取物中的35 kDa蛋白质纯化至同质，并确定其为一种酸性内切木聚糖酶，对燕麦spel木聚糖活性最高。Northern杂交和原位杂交表明，Xyl在四分体小孢子形成单个小孢子后，在花药的绒毡层中特异性表达。Southern杂交和基因拷贝重建研究表明，每个单倍体基因组中只有一个Xyl基因拷贝。对Xyl基因组DNA序列的分析以及对转录本的核糖核酸酶保护研究揭示了启动子区域有许多调控基序，转录本的5'前导区有一个内含子。Xyl转录本有一个562个核苷酸（nt）的5'前导区、一个54 nt的序列编码一个推定的信号肽、一个933 nt的编码序列和一个420 nt的3'非翻译序列。异常长的5'前导区有一个开放阅读框，编码一个推定的175个残基的蛋白质，其序列与一种微生物阿拉伯糖苷酶的序列最相似。玉米木聚糖酶是第一个被证明存在于花粉壁中的酶。文中讨论了它在绒毡层细胞和柱头表面的玉米II型初生细胞壁（以木糖、葡萄糖和阿拉伯糖为主要部分）水解中的可能作用。
