To unravel the function of a protein of interest, it is crucial to asses to what extent it associates via direct interactions or by overlapping expression with other proteins. ROXY1, a land plant‐specific glutaredoxin, exerts a function in Arabidopsis flower development and interacts with TGA transcription factors in the nucleus. We detected a novel ROXY1 function in the root meristem. Root cells that lack chlorophyll reducing plant‐specific background problems that can hamper colocalization 3D microscopy. Thus far, a super‐resolution three‐dimensional stochastic optical reconstruction microscopy (3D‐dSTORM) approach has mainly been applied in animal studies. We established 3D‐dSTORM using the roxy1 mutant complemented with green fluorescence protein‐ROXY1 and investigated its colocalization with three distinct RNAPII isoforms. To quantify the colocalization results, 3D‐dSTORM was coupled with the coordinate‐based colocalization method. Interestingly, ROXY1 proteins colocalize with different RNA polymerase II (RNAPII) isoforms that are active at distinct transcription cycle steps. Our colocalization data provide new insights on nuclear glutaredoxin activities suggesting that ROXY1 is not only required in early transcription initiation events via interaction with transcription factors but likely also participates throughout further transcription processes until late termination steps. Furthermore, we showed the applicability of the combined approaches to detect and quantify responses to altered growth conditions, exemplified by analysis of H₂O₂ treatment, causing a dissociation of ROXY1 and RNAPII isoforms. We envisage that the powerful dual‐color 3D‐dSTORM/coordinate‐based colocalization combination offers plant cell biologists the opportunity to colocalize and quantify root meristem proteins at an increased, unprecedented resolution level <50 nm, which will enable the detection of novel subcellular protein associations and functions.

中文翻译：

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在根分生组织核的整个转录周期中，ROXY1和RNAPII变体的双色3D-dSTORM共定位和定量。

为了揭示目的蛋白质的功能，至关重要的是要评估其通过直接相互作用或与其他蛋白质重叠表达的程度。ROXY1是一种陆地植物特有的谷胱甘肽毒素，在拟南芥花的发育中发挥功能，并与细胞核中的TGA转录因子相互作用。我们在根分生组织中检测到一种新型的ROXY1功能。缺乏叶绿素的根细胞减少了可能会阻碍共定位3D显微镜检查的植物特异性背景问题。到目前为止，超高分辨率三维随机光学重建显微镜（3D-dSTORM）方法已主要应用于动物研究。我们使用roxy1建立了3D‐ dSTORM突变体与绿色荧光蛋白-ROXY1互补，并研究了其与三种不同的RNAPII亚型的共定位。为了量化共定位结果，将3D‐dSTORM与基于坐标的共定位方法结合使用。有趣的是，ROXY1蛋白与在不同转录循环步骤具有活性的不同RNA聚合酶II（RNAPII）同工型共定位。我们的共定位数据提供了关于核戊二醛活性的新见解，表明ROXY1不仅在早期转录起始事件中需要通过与转录因子的相互作用来参与，而且还可能参与进一步的转录过程，直到后期终止步骤为止。此外，我们展示了结合方法的适用性，以检测和量化对变化的生长条件的响应，通过分析H₂ O ₂处理，导致ROXY1和RNAPII亚型解离。我们设想，强大的双色3D-dSTORM /基于坐标的共定位组合为植物细胞生物学家提供了以更高的前所未有的分辨率水平<50 nm共定位和定量根分生组织蛋白的机会，这将使新型亚细胞蛋白的检测成为可能关联和功能。