Molecular characterization of Arabidopsis GAL4/UAS enhancer trap lines identifies novel cell type-specific promoters

Abstract

Cell-type-specific gene expression is essential to distinguish between the numerous cell types of multicellular organism. Therefore, cell-type-specific gene expression is tightly regulated and for most genes RNA transcription is the central point of control. Thus, transcriptional reporters are broadly used markers for cell identity. In Arabidopsis (Arabidopsis thaliana), a recognized standard for cell identities is a collection of GAL4/UAS enhancer trap lines. Yet, while greatly used, very few of them have been molecularly characterized. Here, we have selected a set of 21 frequently used GAL4/UAS enhancer trap lines for detailed characterization of expression pattern and genomic insertion position. We studied their embryonic and postembryonic expression domains and grouped them into three groups (early embryo development, late embryo development, and embryonic root apical meristem lines) based on their dominant expression. We show that some of the analyzed lines are expressed in a domain often broader than the one that is reported. Additionally, we present an overview of the location of the T-DNA inserts of all lines, with one exception. Finally, we demonstrate how the obtained information can be used for generating novel cell-type-specific marker lines and for genotyping enhancer trap lines. The knowledge could therefore support the extensive use of these valuable lines.

Initiation and regulation of vascular tissue identity in theArabidopsisembryo

“…We find that auxin signaling in the central cells of the embryo is indeed required for the complete establishment of the vascular transcription program. This conclusion is supported by an earlier transcriptome analysis, where many vascular genes were downregulated in embryos where the auxin response inhibitor bdl was expressed in the inner cells marked by the Q0990 driver (Radoeva 2016). Interestingly though, several vascular marker genes, including the ZLL gene, persist even upon auxin response inhibition.…”

“…We selected SHR and ATHB8 as early vascular markers in root and leaf development (Baima et al, 2001;Gardiner et al, 2011;Nakajima et al, 2001) . ZLL shows vascular-specific expression in the root and embryo (Radoeva et al, 2016;Tucker et al, 2008;Haseloff, 1999) . WOL, PEAR1 and DOF6 are all associated with cytokinin-responsive growth in the vascular bundle of the root (Mähönen et al, 2000;Miyashima et al, 2019) .…”

“…Since bdl expression in the entire embryo results in early developmental defects (Rademacher et al 2011, Yoshida et al 2014, we employed twocomponent gene activation and selectively expressed bdl in vascular cells using the Q0990-GAL4; UAS-erGFP driver line (Haseloff, 1999). The GAL4 driver in the Q0990 line is inserted near the ZLL gene (Radoeva et al, 2016) and erGFP thus reports ZLL expression. Vascular markers were introduced into the Q0990 background and crossed with a line containing GAL4dependent UAS-bdl (Weijers et al, 2006).…”

“…The reporters for ATHB8 and SHR were previously published (Donner et al 2009, Nakajima et al 2001 . New reporters were generated for WOL and ZLL using primers documented in Table S5, and these reproduce previously described expression patterns (Mähönen et al, 2000;Radoeva et al, 2016) . All newly generated transcriptional and translational reporter constructs (see below) were transformed into the Arabidopsis Col-0 accession.…”

Suspensor-derived somatic embryogenesis in Arabidopsis

“…These include transcriptional regulators such as LEC, BBM, AGL15, RKD, WUS and WOX, and the receptor-like kinase SERK1 (Table S1). Each was misexpressed in the developing suspensor using a two-component GAL4/UAS system from the M0171-GAL4 driver line (Rademacher et al, 2012; Radoeva et al, 2016). This same approach previously led to excessive suspensor cell divisions when the bdl/iaa12 .…”