Fluorescent reporters have facilitated non‐invasive imaging in multiple plant species and thus allowed the analysis of processes ranging from gene expression and protein localization to cellular patterning. However, in rice, a globally important crop and model species, there are relatively few reports of fluorescent proteins being used in leaves. Fluorescence imaging is particularly difficult in the rice leaf blade, likely due to a high degree of light scattering in this tissue. To address this, we investigated approaches to improve deep imaging in mature rice leaf blades. We found that ClearSee treatment, which has previously been used to visualize fluorescent reporters in whole tissues of plants, led to improved imaging in rice. Removing epidermal and subtending mesophyll cell layers was faster than ClearSee and also reduced light scattering such that imaging of fluorescent proteins in deeper leaf layers was possible. To expand the range of fluorescent proteins suitable for imaging in rice, we screened twelve whose spectral profiles spanned most of the visible spectrum. This identified five proteins (mTurquoise2, mNeonGreen, mClover3, mKOκ, and tdTomato) that are robustly expressed and detectable in mesophyll cells of stably transformed plants. Using microparticle bombardment, we show that mTurquoise2 and mNeonGreen can be used for simultaneous multicolor imaging of different subcellular compartments. Overall, we conclude that mTurquoise2, mNeonGreen, mClover3, mKOκ, and tdTomato are suitable for high‐resolution live imaging of rice leaves, both after transient and stable transformation. Along with the rapid microparticle bombardment method, which allows transient transformation of major cell types in the leaf blade, these fluorescent reporters should greatly facilitate the analysis of gene expression and cell biology in rice.

中文翻译：

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用于水稻叶片功能分析的荧光报告基因。

荧光报告基因促进了多种植物物种的非侵入性成像，因此可以分析从基因表达和蛋白质定位到细胞模式的过程。然而，在水稻（全球重要的农作物和模型物种）中，关于叶片中使用荧光蛋白的报道相对较少。水稻叶片中的荧光成像特别困难，这可能是由于该组织中的高度光散射所致。为了解决这个问题，我们研究了改善成熟稻叶片深度成像的方法。我们发现，ClearSee处理（以前已用于可视化植物整个组织中的荧光报告基因）可改善水稻的成像效果。清除表皮和对向的叶肉细胞层的速度比ClearSee快，并且减少了光散射，因此可以在更深的叶层中成像荧光蛋白。为了扩大适用于水稻成像的荧光蛋白的范围，我们筛选了十二种光谱谱涵盖了大部分可见光谱的蛋白。这确定了五种蛋白质（mTurquoise2，mNeonGreen，mClover3，mKOκ和tdTomato）在稳定转化的植物的叶肉细胞中稳定表达并可以检测到。使用微粒轰击，我们表明mTurquoise2和mNeonGreen可用于不同亚细胞区室的同时多色成像。总体而言，我们得出结论，mTurquoise2，mNeonGreen，mClover3，mKOκ和tdTomato适用于水稻叶片的高分辨率实时成像，经过短暂和稳定的转变。随着快速微粒轰击方法的出现，该方法允许叶片中主要细胞类型的瞬时转化，这些荧光报告分子应极大地促进水稻基因表达和细胞生物学的分析。