Abstract：
BACKGROUND: Rosette leaf trichomes of Arabidopsis thaliana have been broadly used to study cell development, cell differentiation and, more recently, cell wall biogenesis. However, trichome-specific biochemical or -omics analyses require a proper separation of trichomes from residual plant tissue. Thus, different strategies were proposed in the past for trichome isolation, which mostly rely on harsh conditions and suffer from low yield, thereby limiting the spectrum of downstream analyses.
RESULTS: To take trichome-leaf separation to the next level, we revised a previously proposed method for isolating A. thaliana trichomes by optimizing the mechanical and biochemical specifications for trichome release. We additionally introduced a density gradient centrifugation step to remove residual plant debris. We found that prolonged, yet mild seedling agitation increases the overall trichome yield by more than 60% compared to the original protocol. We noticed that subsequent density gradient centrifugation further visually enhances trichome purity, which may be advantageous for downstream analyses. Gene expression analysis by quantitative reverse transcriptase-polymerase chain reaction validated a substantial enrichment upon purification of trichomes by density gradient centrifugation. Histochemical and biochemical investigation of trichome cell wall composition indicated that unlike the original protocol gentle agitation during trichome release largely preserves trichome integrity. We used enriched and density gradient-purified trichomes for proteomic analysis in comparison to trichome-depleted leaf samples and present a comprehensive reference data set of trichome-resident and -enriched proteins. Collectively we identified 223 proteins that are highly enriched in trichomes as compared to trichome-depleted leaves. We further demonstrate that the procedure can be applied to retrieve diverse glandular and non-glandular trichome types from other plant species.
CONCLUSIONS: We provide an advanced method for the isolation of A. thaliana leaf trichomes that outcompetes previous procedures regarding yield and purity. Due to the large amount of high-quality trichomes our method enabled profound insights into the so far largely unexplored A. thaliana trichome proteome. We anticipate that our protocol will be of use for a variety of downstream analyses, which are expected to shed further light on the biology of leaf trichomes in A. thaliana and possibly other plant species.
RESULTS: To take trichome-leaf separation to the next level, we revised a previously proposed method for isolating A. thaliana trichomes by optimizing the mechanical and biochemical specifications for trichome release. We additionally introduced a density gradient centrifugation step to remove residual plant debris. We found that prolonged, yet mild seedling agitation increases the overall trichome yield by more than 60% compared to the original protocol. We noticed that subsequent density gradient centrifugation further visually enhances trichome purity, which may be advantageous for downstream analyses. Gene expression analysis by quantitative reverse transcriptase-polymerase chain reaction validated a substantial enrichment upon purification of trichomes by density gradient centrifugation. Histochemical and biochemical investigation of trichome cell wall composition indicated that unlike the original protocol gentle agitation during trichome release largely preserves trichome integrity. We used enriched and density gradient-purified trichomes for proteomic analysis in comparison to trichome-depleted leaf samples and present a comprehensive reference data set of trichome-resident and -enriched proteins. Collectively we identified 223 proteins that are highly enriched in trichomes as compared to trichome-depleted leaves. We further demonstrate that the procedure can be applied to retrieve diverse glandular and non-glandular trichome types from other plant species.
CONCLUSIONS: We provide an advanced method for the isolation of A. thaliana leaf trichomes that outcompetes previous procedures regarding yield and purity. Due to the large amount of high-quality trichomes our method enabled profound insights into the so far largely unexplored A. thaliana trichome proteome. We anticipate that our protocol will be of use for a variety of downstream analyses, which are expected to shed further light on the biology of leaf trichomes in A. thaliana and possibly other plant species.
摘要：
背景：拟南芥的玫瑰花叶毛状体已被广泛用于研究细胞发育，细胞分化和，最近,细胞壁生物发生。然而,特定于毛状体的生化或组学分析需要从残留的植物组织中适当分离毛状体。因此，过去提出了不同的毛状体隔离策略，主要依赖恶劣的条件，产量低，从而限制了下游分析的范围。
结果：为了使毛状体-叶分离更上一层楼，我们通过优化毛状体释放的机械和生化规范，修改了先前提出的分离拟南芥毛状体的方法。我们另外引入了密度梯度离心步骤以去除残留的植物碎片。我们发现，然而，与原始方案相比，温和的幼苗搅拌使毛状体的整体产量增加了60％以上。我们注意到，随后的密度梯度离心进一步在视觉上提高了毛状体的纯度，这对于下游分析可能是有利的。通过定量逆转录酶-聚合酶链反应进行的基因表达分析验证了通过密度梯度离心纯化毛状体时的大量富集。毛状体细胞壁组成的组织化学和生化研究表明，与原始方案不同，毛状体释放过程中的温和搅动在很大程度上保留了毛状体的完整性。我们使用富集和密度梯度纯化的毛状体进行蛋白质组学分析，与毛状体耗尽的叶片样品进行比较，并提供了毛状体驻留和富集蛋白的综合参考数据集。总的来说，我们鉴定了223种蛋白质，与毛状体耗尽的叶子相比，这些蛋白质在毛状体中高度富集。我们进一步证明，该程序可用于从其他植物物种中检索各种腺状和非腺状毛状体类型。
结论：我们提供了一种分离拟南芥叶毛状体的先进方法，该方法在产量和纯度方面优于以前的程序。由于大量的高质量毛状体，我们的方法使人们对迄今为止尚未开发的A.thaliana毛状体蛋白质组具有深刻的见解。我们预计我们的协议将用于各种下游分析，有望进一步阐明拟南芥和其他植物物种的叶毛状体的生物学。
结果：为了使毛状体-叶分离更上一层楼，我们通过优化毛状体释放的机械和生化规范，修改了先前提出的分离拟南芥毛状体的方法。我们另外引入了密度梯度离心步骤以去除残留的植物碎片。我们发现，然而，与原始方案相比，温和的幼苗搅拌使毛状体的整体产量增加了60％以上。我们注意到，随后的密度梯度离心进一步在视觉上提高了毛状体的纯度，这对于下游分析可能是有利的。通过定量逆转录酶-聚合酶链反应进行的基因表达分析验证了通过密度梯度离心纯化毛状体时的大量富集。毛状体细胞壁组成的组织化学和生化研究表明，与原始方案不同，毛状体释放过程中的温和搅动在很大程度上保留了毛状体的完整性。我们使用富集和密度梯度纯化的毛状体进行蛋白质组学分析，与毛状体耗尽的叶片样品进行比较，并提供了毛状体驻留和富集蛋白的综合参考数据集。总的来说，我们鉴定了223种蛋白质，与毛状体耗尽的叶子相比，这些蛋白质在毛状体中高度富集。我们进一步证明，该程序可用于从其他植物物种中检索各种腺状和非腺状毛状体类型。
结论：我们提供了一种分离拟南芥叶毛状体的先进方法，该方法在产量和纯度方面优于以前的程序。由于大量的高质量毛状体，我们的方法使人们对迄今为止尚未开发的A.thaliana毛状体蛋白质组具有深刻的见解。我们预计我们的协议将用于各种下游分析，有望进一步阐明拟南芥和其他植物物种的叶毛状体的生物学。
