New pOp/LhG4 vectors for stringent glucocorticoid‐dependent transgene expression in Arabidopsis

Abstract

These authors contributed equally to this work. SummaryTo facilitate glucocorticoid-inducible transgene expression from the pOp promoter in Arabidopsis the ligandbinding domain of a rat glucocorticoid receptor (GR LBD) was fused to the amino terminus of the synthetic transcription factor LhG4 to generate LhGR-N. Fusions bearing the GR LBD at other positions in LhG4 exhibited incomplete repression or inefficient induction. LhGR-N was stringently repressed in the absence of exogenous glucocorticoid but was fully activated by addition of 2 lM dexamethasone which resulted in 1000-fold increase in GUS reporter activity. Half maximal induction was achieved with 0.2 lM dexamethasone. Reporter transcripts were detectable within 2 h of dexamethasone application and peaked 4-10 h later. Neither LhGR-N nor dexamethasone affected seedling development although ethanol retarded development when used as a solvent for dexamethasone. The efficiency of the pOp target promoter was improved 10-to 20-fold by incorporating six copies of the ideal lac operator with sufficient inter-operator spacing to allow simultaneous occupancy. Introduction of the TMV X sequence into the 5¢UTR resulted in a further 10-fold increase in dexamethasone-inducible reporter activity and an increase in the induction factor to 10 4 . Although promoters containing the TMV X sequence exhibited slightly increased basal expression levels in the absence of dexamethasone, stringent regulation of the cytokinin biosynthetic gene ipt was achieved with all promoters. Despite the severity of the induced ipt phenotypes, transcripts for the KNOX homoeodomain transcription factors BREVIPEDICELLUS and SHOOTMERISTEMLESS were not significantly increased within 48 h of dexamethasone application to seedlings.

Targeted manipulation of leaf form via local growth repression

“…Although the depth of sinus indentation was clearly increased towards the base of the leaves, a smaller change was observed towards the leaf tip, suggesting that the basic pattern of lobbing/serration was not altered by these manipulations. The pOp synthetic promoter used in these experiments (Craft et al, 2005;Samalova et al, 2005) resulted in simultaneous transcription of AtKRP1 and a GUS reporter gene after Dex induction, allowing verification that target gene expression was localized to the expected region of the leaf flank ( Figure S1d-g). Analysis of leaf histology showed that the cells in the region of CUC2-directed AtKRP1 expression were greatly enlarged relative to adjacent cells (Figure 2e-g) and relative to cells in equivalent positions in wild-type leaves (Figure 2c).…”

“…The amplified fragment was subsequently cloned into the pENTR-DTOPO vector, and then recombined into the pBIN-LRLhGR2 vector using LR clonase (Invitrogen). For the second component of the CUC2>>KRP1 plants, SalI and BamHI restriction sites were added to a KRP1 109 fragment using primers 5¢-acgcGTCGACATGGAATTTGAATCGGCGGTTAAAG-3¢ and 5¢-acgcGTCGACATGGTGAGAAAATATAGAAAAGCT-3¢, and, after restriction digest, fragments were ligated into a pV-TOP vector (Craft et al, 2005) using T4 DNA ligase (Promega). After transformation into Arabidopsis (Clough and Bent, 1998), homozygous progeny for each component (pBIN-CUC2-LR-LhGR2 and pV-TOP-KRP1) were obtained and crossed.…”

“…We therefore decided to use expression of AtKRP1 as a tool to repress growth in a targeted fashion during leaf development. To regulate the spatial/temporal expression pattern of this growth repressor, we used established inducible promoter and enhancer trap systems (Craft et al, 2005;Laplaze et al, 2005). These systems provide a means of generating novel patterns of AtKRP1 expression at specific time points in leaf development, thus allowing us to observe the effect of new patterns of growth repression on leaf form.…”

GLK Transcription Factors Coordinate Expression of the Photosynthetic Apparatus in Arabidopsis

“…The aim was to measure transcriptome changes following induction. A twocomponent glucocorticoid-inducible system was used to drive GLK1 or GLK2 expression following treatment with dexamethasone (DEX) (Craft et al, 2005). Briefly, the GLK1 and GLK2 coding sequences were cloned behind a chimeric promoter consisting of a minimal cauliflower mosaic virus 35S promoter and six ideal lac operator sequences, termed pOp6 (Kannangara et al, 2007).…”

“…The activator line LhGR-N(4c), carrying a single copy of the 35S:LhGR-N construct (Craft et al, 2005), was crossed with the glk1 glk2 mutant. The dark-green F1 progeny were selected on kanamycin and selfed.…”