Dual‐color 3D‐dSTORM colocalization and quantification of ROXY1 and RNAPII variants throughout the transcription cycle in root meristem nuclei. (5th October 2020)

• Record Type: Journal Article
• Authors: Maß, Lucia; Holtmannspötter, Michael; Zachgo, Sabine
• Is Part Of: Plant journal. Volume 104:Number 5(2020)
• Page Start: 1423
• Page End: 1436
• Publication Date: 2020-10-05
• Subjects: ROXY1 -- RNAPII -- super‐resolution microscopy -- dual‐color 3D‐dSTORM -- colocalization -- transcription cycle -- root meristem -- technical advance; Plant molecular biology -- Periodicals; Plant cells and tissues -- Periodicals; Botany -- Periodicals; 580
• DOI: 10.1111/tpj.14986
• Languages: English
• ISSNs: 0960-7412

Abstract

SUMMARY: 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 H2 O2 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. Significance Statement: This paper presents the application of 3D‐dSTORM for the detection of proteins in Arabidopsis root meristem nuclei at a resolution <50 nm, thus facilitating single molecule imaging in plant cells. The combination of dual‐color 3D‐dSTORM studies with a coordinate‐based colocalization approach for signal quantification revealed the colocalization of ROXY1 with distinct RNA polymerase II isoforms during the transcription cycle, which suggest novel functions for land plant‐specific ROXY glutaredoxins.