Reporters for sensitive and quantitative measurement of auxin response

Abstract

The visualization of hormonal signaling input and output is key to understanding how multicellular development is regulated. The plant signaling molecule auxin triggers many growth and developmental responses, but current tools lack the sensitivity or precision to visualize these. We developed a set of fluorescent reporters that allow sensitive and semiquantitative readout of auxin responses at cellular resolution in Arabidopsis thaliana. These generic tools are suitable for any transformable plant species.

Figure 1. DR5v2 sensitively reports auxin response

(a) Schematic of DR5v2-ntdTomato/DR5-n3GFP double reporter. 10 repeats of either reverse TGTCTC (DR5) or TGTCGG (DR5v2) are positioned upstream of a minimal promoter and either nuclear 3×eGFP (n3GFP) or nuclear tandem Tomato (ntdTomato). LB/RB, Left/Right Border; DHFR, Methotrexate resistance gene. (b-j) DR5v2 (red) and DR5 (Green) activity in early globular (b) and heart stage (c) embryos, root tip (d, longitudinal section; e, transverse section along plane at arrowhead in d; px, protoxylem; mx, metaxylem; peri, pericycle), lateral root cap (f), root epidermis (g, here shown in a DR5v2::n3eGFP root), SAM (h) and young leaf (i). (j,k) Relative qRT-PCR GFP and tdTomato transcript level in DR5v2-ntdTomato/DR5-n3GFP seedlings after 12 hour pre-treatment with 10 μM NPA followed by 2 hour treatment with auxin (IAA) concentrations as indicated (j), or treated with 1 μM IAA for the indicated times without NPA pre-treatment (k). Expression in mock treatments in are set to 1. Bars indicate standard error from the mean (n= 3). Asterisks (*) indicate significantly different expression compared to untreated control, while number signs (#) indicate significant difference between DR5 and DR5v2 (Two-tailed t-test; p<0.05). (l) Visualization of DR5v2 (red)-DR5 (Green) double reporter activity in root tips after 12-hour co-treatment of 10 μM NPA with indicated IAA concentrations. Scale bar in panels (b-i, l) is 10 μm.

Figure 2. R2D2, a semi-quantitative and rapid auxin input reporter

(a) Schematic of R2D2. LB/RB, Left/Right Border; DHFR, Methothrexate resistance gene. (b-k) ntdTomato (red) and n3×Venus (green) fluorescence signal overlays (b-e, h-j) and inverse n3×Venus/ ntdTomato signal ratio (f,g,k; false color) in pre-globular (b) and heart stage (c) embryos, root tip (longitudinal section in d,e; radial section in e,g; detail in j,k), young leaf (h) and SAM (i). Note the descending gradient of auxin input in RAM in (j,k). (l) Successive images of R2D2 root tips treated with 1 μM IAA for the indicated time. (m) Whole-frame quantification of inverse n3×Venus/ ntdTomato signal ratio after treatment with 1 μM IAA and untreated mock control. Bars indicate standard error from the mean (n=3). Scale bar in panels (b-l) is 10 μm.