Microgametogenesis is a complex process that involves numerous well-coordinated cell activities, ending with the production of pollen grains. Pollen development has been studied at the cytological level in Arabidopsis and other plant species, where its temporal time course has been defined. However, the molecular mechanism underlying this process is still unclear, since a relative small number of genes and/or processes have been identified as essential for pollen development. We have designed a methodology to select candidate genes for functional analysis, based on transcriptomic data obtained from different stages of pollen development. From our analyses, we selected At2g22950 as a candidate gene; this gene encodes a protein belonging to the auto-regulated Ca(2+)-ATPase family, ACA7. Microarray data indicate that ACA7 is expressed exclusively in developing pollen grains, with the highest level of mRNA at the time of the second pollen mitosis. Our RT-PCR experiments showed that ACA7 mRNA is detected exclusively in developing flowers. Confocal microscopy experiments showed a plasma membrane localization for the recombinant GFP:ACA7 protein. We identified two different insertional mutant lines, aca7-1 and aca7-2; plants from both mutant lines displayed a normal vegetative development but showed large amounts of dead pollen grains in mature flowers assayed by Alexander's staining. Histological analysis indicated that abnormalities are detected after the first pollen mitosis and we found a strong correlation between ACA7 mRNA accumulation and the severity of the phenotype. Our results indicate that ACA7 is a plasma membrane protein that has an important role during pollen development, possibly through regulation of Ca(2+) homeostasis.

中文翻译：

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拟南芥 ACA7 编码一种推定的自动调节 Ca(2+)-ATP 酶，是正常花粉发育所必需的。


小配子发生是一个复杂的过程，涉及许多协调良好的细胞活动，最终产生花粉粒。人们在细胞学水平上对拟南芥和其他植物物种的花粉发育进行了研究，并确定了其时间进程。然而，该过程背后的分子机制仍不清楚，因为相对少量的基因和/或过程已被确定为花粉发育所必需的。我们设计了一种方法，根据从花粉发育的不同阶段获得的转录组数据来选择候选基因进行功能分析。根据我们的分析，我们选择At2g22950作为候选基因；该基因编码属于自动调节 Ca(2+)-ATP 酶家族 ACA7 的蛋白质。微阵列数据表明，ACA7 仅在发育中的花粉粒中表达，在第二次花粉有丝分裂时 mRNA 水平最高。我们的 RT-PCR 实验表明，ACA7 mRNA 仅在发育中的花中检测到。共聚焦显微镜实验显示重组 GFP:ACA7 蛋白在质膜上定位。我们鉴定了两种不同的插入突变株系：aca7-1 和 aca7-2；两个突变株系的植物都表现出正常的营养发育，但通过亚历山大染色分析，在成熟的花朵中显示出大量死亡的花粉粒。组织学分析表明，在第一次花粉有丝分裂后检测到异常，我们发现 ACA7 mRNA 积累与表型严重程度之间存在很强的相关性。我们的结果表明，ACA7 是一种质膜蛋白，在花粉发育过程中发挥重要作用，可能是通过调节 Ca(2+) 稳态来实现的。