Department of Plant and Microbial Biology, University of California Berkeley, Berkeley, CA, USA.
Biophysics Graduate Group, University of California Berkeley, Berkeley, CA, USA.
Nat Plants. 2021 Aug;7(8):1037-1049. doi: 10.1038/s41477-021-00976-0. Epub 2021 Aug 9.
The responses of plants to their environment are often dependent on the spatiotemporal dynamics of transcriptional regulation. While live-imaging tools have been used extensively to quantitatively capture rapid transcriptional dynamics in living animal cells, the lack of implementation of these technologies in plants has limited concomitant quantitative studies in this kingdom. Here, we applied the PP7 and MS2 RNA-labelling technologies for the quantitative imaging of RNA polymerase II activity dynamics in single cells of living plants as they respond to experimental treatments. Using this technology, we counted nascent RNA transcripts in real time in Nicotiana benthamiana (tobacco) and Arabidopsis thaliana. Examination of heat shock reporters revealed that plant tissues respond to external signals by modulating the proportion of cells that switch from an undetectable basal state to a high-transcription state, instead of modulating the rate of transcription across all cells in a graded fashion. This switch-like behaviour, combined with cell-to-cell variability in transcription rate, results in mRNA production variability spanning three orders of magnitude. We determined that cellular heterogeneity stems mainly from stochasticity intrinsic to individual alleles instead of variability in cellular composition. Together, our results demonstrate that it is now possible to quantitatively study the dynamics of transcriptional programs in single cells of living plants.
植物对环境的反应通常依赖于转录调控的时空动态。虽然活体成像工具已被广泛用于定量捕获活体动物细胞中快速的转录动态，但这些技术在植物中的应用有限，限制了该领域的相应定量研究。在这里，我们应用 PP7 和 MS2 RNA 标记技术，对活体植物单细胞中 RNA 聚合酶 II 活性动态进行定量成像，以响应实验处理。使用这项技术，我们实时计数了烟草原生质体（烟草）和拟南芥中新生 RNA 转录本的数量。对热激报告基因的检测表明，植物组织通过调节从不可检测的基础状态切换到高转录状态的细胞比例来对外界信号做出反应，而不是以分级的方式调节所有细胞的转录速率。这种开关样行为，加上转录率的细胞间可变性，导致 mRNA 产生的可变性跨越三个数量级。我们确定细胞异质性主要源于单个等位基因固有的随机性，而不是细胞组成的可变性。总之，我们的结果表明，现在可以定量研究活体植物单细胞中转录程序的动态。
