Figure 1.
In vitro-translated PRN1 has quercetinase activity.
A. in-vitro-translated PRN1 possesses quercetinase activity. In vitro-translated PRN1 was incubated with a final concentration of 10 μM quercetin in a 500 μL total reaction volume, or as a control, 10 μM quercetin alone, in buffer as described [12]. Reactions were carried out in darkness in a 26°C water bath for 15 min. After incubation at 26°C, absorption spectra of quercetin alone (Quercetin only), Quercetin+PRN1, Quercetin+protein extract, or PRN1 extract only were determined using a Perkin Elmer scanning spectrophotometer (Perkin Elmer; Waltham, MA). n = 3, SD shown. B. The conformational status of GPA1 affects the quercetinase ability of PRN1. In vitro-translated GPA1 was pre-incubated with non-hydrolyzable analogs (either GTPγS or GDPβS) overnight at 10°C, then was incubated with PRN1, then (PRN1+GPA1+GTPγS or PRN1+GPA1+GDPβS) was added to buffer+quercetin to assess the effects on quercetinase activity as shown. Absorption spectra of quercetin alone (Quercetin only) also shown. n = 3, SD shown.
Figure 2.
prn1 mutants accumulate excess quercetin, and can survive UV-C irradiation that kills WT.
A. prn1 mutants accumulate more quercetin compared to WT seedlings. 6-d-old dark-grown seedlings were treated with a brief pulse of UV (317 nm), returned to darkness, then harvested 6 h later to determine the total extractable quercetin (Q) or kaempferol (K) in WT or prn1 seedlings (WT-Q; prn1-Q; WT-K; prn1-K). The levels of Q and K are indicated for prn1 and WT and SD are shown. n = 4 B & C. prn1 mutants survive a UV-C radiation treatment that kills WT seedlings. Seedlings grown in complete darkness as described in methods. UV-C treatments were administered on d 6 as described [30], with 4 min (B) or 8 min (C) of 254 nm (UV-C) radiation. Seedlings were returned to complete darkness for 24 h. UV-C killing doses for adt3 or WT were determined by prior ‘titration’ experimentation [30]. Seedlings were photographed from the side. n = 4. D. prn1 seedlings transformed with PRN1::PRN1-GFP construct are killed by 8 min of UV-C, similar to WT. Seeds (30) of WT, prn1 or prn1 transformed with PRN1::PRN1-GFP (PRN1::PRN1-GFP prn1) were sown, then seedlings grown in complete darkness. UV-C treatments were administered on d 6 as described [30], with an 8 min dose of 254 nm (UV-C) radiation. Seedlings were returned to complete darkness for 24 h. Seedlings were photographed from the side. n = 4.
Figure 3.
3-d-old light-grown prn1 mutants exhibit abnormal light-grown shoot orientation phenotype.
Three (72 h)-d-old white light-grown seedlings were photographed from the side using a dissecting microscope to compare WT (A–B) and prn1 mutant (C–E) seedlings. Images are representative of the range of orientations observed. Scale bars each represent 1.0 mm. Hypocotyl angles were measured for individual seedlings in reference to the horizontal phytatray surface (where vertical = 90°) and mean angles were calculated (F). Means are shown by lines (solid for WT; dotted for prn1) and SEMs are represented by shaded triangles. n = 65. SEM shown.
Figure 4.
Subcellular localization of PRN1 in dark-grown seedlings.
Constructs and transgenic plants are described in methods. 6-d-old dark-grown prn1 whole seedlings transformed with PRN1::PRN1-GFP (T3) or WT seedlings transformed with 35S::PRN1-GFP were fixed, stained with DAPI, mounted on slides, then photographed on a spinning disk confocal using steady state lasers 405 nm, 488 nm, and 561 nm. All images shown are from the cotyledon epidermis layer. Panel rows are indicated by wavelength, with individual channels in black and white. In the merge, 405 nm (DAPI) is false-colored blue, 488 nm (GFP) is false-colored green, and 561 nm is false-colored red (Red). Images are representative with no alteration of the fluorescence within the image field, and images represent an optical section of 1 μm thickness. The column heading indicates the different seedling lineages. Yellow boxes on the figure indicate areas of interest and show an enlargement. Red arrows indicate DAPI stain in nucleus. White arrows indicate GFP accumulation in 488 nm. Untransformed WT and prn1 seedlings are also shown on the figure. Scale bars each represent 25 μm. n = 4 biological replicates, with at least 12–20 individual seedlings viewed per replicate; images are representative.
Figure 5.
Enlargement and z-slices of region of interest in seedling cotyledon epidermis: prn1 transformed with PRN1::PRN1-GFP.
Microscopy and techniques are the same as described for Figure 4. Each row shows 5 consecutive z-slices. The red “n” indicates a nucleus (DAPI stained). GFP expression of interest is indicated by white arrows. Scale bar represents 10 μm.
Figure 6.
Enlargement and z-slices of region of interest in seedling cotyledon epidermis: WT transformed with 35S::PRN1-GFP.
Microscopy and techniques are the same as described for Figure 4. Each row shows 5 consecutive z-slices. The red “n” indicates a nucleus (DAPI stained). GFP expression of interest is indicated by white arrows. There is expression in the rest of the cell, largely diffuse. Scale bar represents 10 μm.