Rapid initiation of Arabidopsis pollination requires the oleosin-domain protein GRP17
Rapid initiation of Arabidopsis pollination requires the oleosin-domain protein GRP17
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DOI：
10.1038/35000084
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发表时间：
2000-02-01
影响因子：
21.3
通讯作者：
Preuss, D
中科院分区：
文献类型：
--
作者：
Mayfield, JA;Preuss, D
* Department of Molecular Genetics and Cell Biology，The University of Chicago，Chicago，Illinois 60637，USA †电子邮件：dpreuss@midway。乌奇哥。在高等植物配子融合之前，发生了一系列令人印象深刻的细胞相互作用，即花粉对雌性柱头细胞的种特异性粘附、干燥花粉的调节水合作用、花粉管的萌发以及花粉管向胚珠的引导生长。控制花粉水化的能力为植物提供了早期识别和排除不需要的花粉的机会。水化的开始和水的快速转移由富含脂质的细胞外花粉衣介导。两条线的证据提示，水合作用只需要脂质：外源脂质促进花粉水合作用，即使在不相容的表面2;和所有以前报道的水合突变体有缺陷，破坏脂质biosynthesis3，4。然而，添加的脂质可能会绕过重要的调节或信号传导事件，并且水合突变体表现出整个花粉外壳的多效性降解，使得单个分子难以分析（图1）。在这里，我们描述了一个突变，grp17 - 1，删除一个单一的油质蛋白结构域的蛋白质从拟南芥花粉衣。这种突变损害花粉的水合作用和竞争能力，描绘了蛋白质在水合作用中的重要性。(Note两个独立的拟南芥基因之前被命名为GRP 7。在咨询了拟南芥共同体标准的顾问后，我们将油质蛋白结构域蛋白称为GRP17。助理拟南芥花粉涂层含有少量蛋白质（图1a）。最丰富的迁移在相对分子质量为49,000（先生49K），并包括21%的总外壳蛋白。对来自该条带的羧基末端和内部肽进行微测序，明确鉴定了GRP17，这是一种先前在寻找富含甘氨酸的蛋白质时发现的蛋白质5。与GRP 17 DNA序列相关的多态性或GRP 17蛋白的迁移率映射到5号染色体上的相同间隔。此外，GRP17表达是雄性特异性的，与花粉衣中的定位一致5。GRP17具有保守的油质蛋白结构域和高度发散的带电结构域。种子定位oleosins调节油体的大小，防止脂质聚集在种子发育6; oleosins的能力，管理脂质特性，使他们有趣的候选人的调节花粉hydration.We确定了一种植物含有T-DNA元件的GRP17基因，这种病变（grp17 - 1）导致删除氨基酸146 - 150和插入一个提前终止密码子。该杂合植株为Wassilewskija（Ws-2）生态型，形态正常，完全可育。来自该植物的后代以3：1的比例分离由T-DNA盒赋予的卡那霉素抗性（815个非抗性：2，358个卡那霉素抗性; χ 2：0.5> P> 0.1）。自花授粉的杂合子以预期的孟德尔比率产生健康后代（14 +/+：29 +/-：12-/-; χ 2：0.9> P> 0.5）。北方印迹和逆转录聚合酶链反应（RT-PCR）均未检测到突变花中的GRP17信使RNA（数据未显示）;此外，突变的pol-RNA聚合酶链反应（pol-RNA聚合酶链反应）也未检测到突变花中的GRP17信使RNA。
* Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, Illinois 60637, USA† e-mail: dpreuss@ midway. uchicago. edu n impressive set of cellular interactions occurs before the fusion of higher-plant gametes, namely species-specific adhesion of pollen to female stigma cells, regulated hydration of desiccated pollen, germination of a pollen tube, and guided growth of the pollen tube to the ovules. The ability to control pollen hydration affords the plant an early opportunity to recognize and exclude undesirable pollen1. The onset of hydration and rapid transfer of water are mediated by the lipid-rich, extracellular pollen coat. Two lines of evidence hint that hydration requires only lipids: exogenous lipids facilitate pollen hydration, even on incompatible surfaces2; and all previously reported hydration mutants have defects that disrupt lipid biosynthesis3, 4. However, added lipids may bypass important regulatory or signalling events, and the hydration mutants exhibit a pleiotropic degradation of the entire pollen coat, rendering individual molecules difficult to analyse (Fig. 1). Here we describe a mutation, grp17-1, that removes a single oleosin-domain protein from the Arabidopsis thaliana pollen coat. This mutation impairs pollen hydration and competitive ability, delineating the importance of proteins in hydration.(Note that two separate Arabidopsis genes were previously given the name GRP7. Having consulted with advisors on Community Standards for Arabidopsis, we refer to the oleosin-domain protein as GRP17.) The A. thaliana pollen coating contains a small number of proteins (Fig. 1a). The most abundant migrated at a relative molecular mass of 49,000 (Mr 49K) and comprised 21% of the total coat protein. Microsequencing of carboxy-terminal and internal peptides derived from this band unambiguously identified GRP17, a protein previously discovered in a search for glycine-rich proteins5. Polymorphisms associated with the GRP17 DNA sequence or mobility of the GRP17 protein mapped to the same interval on chromosome 5. Further, GRP17 expression is male-specific, consistent with a localization in the pollen coat5. GRP17 has a conserved oleosin domain and a highly divergent charged domain. Seed-localized oleosins regulate oil-body size and prevent lipid aggregation during seed development6; the ability of oleosins to govern lipid properties makes them intriguing candidates for the regulation of pollen hydration.We identified a plant containing a T-DNA element in the GRP17 gene; this lesion (grp17-1) resulted in the removal of amino acids 146–150 and inserted a premature stop codon. This heterozygous plant, of the Wassilewskija (Ws-2) ecotype, was morphologically normal and fully fertile. Progeny from this plant segregated kanamycin resistance, conferred by the T-DNA cassette, in a 3: 1 ratio (815 non-resistant: 2,358 kanamycin-resistant; χ2: 0.5> P> 0.1). Selfpollinated heterozygotes produced healthy offspring in the expected mendelian ratios (14+/+: 29+/–: 12–/–; χ2: 0.9> P> 0.5). Neither northern blots nor reverse transcription with polymerase chain reaction (RT-PCR) detected GRP17 messenger RNA in the mutant flowers (data not shown); furthermore, the mutant pol-