Root-localized phytochrome chromophore synthesis is required for photoregulation of root elongation and impacts root sensitivity to jasmonic acid in Arabidopsis

Abstract

Plants exhibit organ- and tissue-specific light responses. To explore the molecular basis of spatial-specific phytochrome-regulated responses, a transgenic approach for regulating the synthesis and accumulation of the phytochrome chromophore phytochromobilin (PΦB) was employed. In prior experiments, transgenic expression of the BILIVERDIN REDUCTASE (BVR) gene was used to metabolically inactivate biliverdin IXα, a key precursor in the biosynthesis of PΦB, and thereby render cells accumulating BVR phytochrome deficient. Here, we report analyses of transgenic Arabidopsis (Arabidopsis thaliana) lines with distinct patterns of BVR accumulation dependent upon constitutive or tissue-specific, promoter-driven BVR expression that have resulted in insights on a correlation between root-localized BVR accumulation and photoregulation of root elongation. Plants with BVR accumulation in roots and a PΦB-deficient elongated hypocotyl2 (hy2-1) mutant exhibit roots that are longer than those of wild-type plants under white illumination. Additional analyses of a line with root-specific BVR accumulation generated using a GAL4-dependent bipartite enhancer-trap system confirmed that PΦB or phytochromes localized in roots directly impact light-dependent root elongation under white, blue, and red illumination. Additionally, roots of plants with constitutive plastid-localized or root-specific cytosolic BVR accumulation, as well as phytochrome chromophore-deficient hy1-1 and hy2-1 mutants, exhibit reduced sensitivity to the plant hormone jasmonic acid (JA) in JA-dependent root inhibition assays, similar to the response observed for the JA-insensitive mutants jar1 and myc2. Our analyses of lines with root-localized phytochrome deficiency or root-specific phytochrome depletion have provided novel insights into the roles of root-specific PΦB, or phytochromes themselves, in the photoregulation of root development and root sensitivity to JA.

Figure 1.

Whole-mount immunolocalization of BVR protein accumulation in roots of wild-type and promoter-driven BVR seedlings. No-0 wild-type (A and F), 35S::pBVR3 (B and G), 35S::cBVR1 (C and H) CAB3::pBVR2 (D and I), and MERI5::pBVR1 (E and J) seedlings were grown on Phytablend medium containing 1% (w/v) Suc for approximately 4 d at 22°C under Wc illumination of 100 μmol m⁻² s⁻¹. Seedlings were incubated with anti-BVR primary antibody at a 1:4,000 dilution, except for 1:3,000 dilution for 35S::cBVR1. The top row shows fluorescence images and the bottom row shows DIC images for each seedling. Each image is a representative slice from a Z-series with 0.5-μm interval size and was captured using 543-nm laser excitation with a 20× lens objective. Fluorescence images were collected using a 560- to 615-nm band-pass filter. Bars = 50 μm.

Figure 2.

Photomorphogenesis and root elongation responses of the wild type, phytochrome chromophore-deficient, and JA-insensitive mutants grown under Wc illumination. No-0 wild-type (WT), 35S::pBVR3, 35S::cBVR1, CAB3::pBVR2 (CAB-2), MERI5::pBVR1 (MERI-1), C24 wild-type, F3 seedlings of a M0062 × UASBVR cross (M0062/UASBVR), C20 wild-type, hy1-1, hy2-1, Col-0 wild-type, jar1, and myc2 lines were grown at 22°C on Phytablend medium containing 1% (w/v) Suc with no added MeJA for 10 d at 22°C under Wc illumination of 100 μmol m⁻² s⁻¹. A, Images of seedlings. Bars = 1 cm. B, Bars represent means ± sd of root lengths in mm (n ≥ 10 for each of six independent experiments). For statistical significance tests, comparisons were made relative to cognate wild-type lines: ^a P < 0.0001, ^b P < 0.01, ^c P < 0.05. For information on the range of seedling lengths observed for each line, see summarized frequency distribution data (Supplemental Table S1).

Figure 3.

Whole-mount immunolocalization of BVR protein accumulation in roots of wild-type and enhancer-trap BVR seedlings. C24 wild-type seedlings (WT; A–D) and F3 seedlings of a M0062 × UASBVR cross (M0062/UASBVR; E–H) were grown on Phytablend medium containing 1% (w/v) Suc for approximately 4 d at 22°C under Wc illumination of 100 μmol m⁻² s⁻¹. Fluorescence images of seedlings incubated without (A and E) or with (C and G) anti-BVR primary antibody (Ab) at a 1:2,000 dilution are shown. DIC images (B, D, F, and H) are shown for each seedling. Each image is a representative slice from a Z-series with 0.5-μm interval size, captured using 543-nm laser excitation with a 20× lens objective. Fluorescence images were collected using a 560- to 615-nm band-pass filter. Bars = 50 μm.

Figure 4.

Light-dependent root elongation responses of the wild type, phytochrome chromophore-deficient, and phytochrome apoprotein mutants. No-0 wild-type (WT), 35S::pBVR3, 35S::cBVR1, C24 wild-type, F3 seedlings of a M0062 × UASBVR cross (M0062/UASBVR), C20 wild-type, hy1-1, hy2-1, Col-0 wild-type, phyA, and phyB lines were grown on Phytablend medium containing 1% (w/v) Suc for 10 d at 22°C under Bc light of 30 μmol m⁻² s⁻¹, Rc light of 50 μmol m⁻² s⁻¹, and FRc light of 10 μmol m⁻² s⁻¹. Bars represent means ± sd of root lengths in mm (n ≥ 10 for each of three independent experiments). For statistical significance tests, comparisons were made relative to cognate wild-type lines: ^a P < 0.001, ^b P < 0.01, ^c P < 0.05.

Figure 5.

Photomorphogenesis and mean hypocotyl lengths of wild-type and BVR-expressing seedlings. No-0 wild-type (WT), 35S::cBVR1, C24 wild-type, and M0062/UASBVR lines were grown on Phytablend medium containing 1% (w/v) Suc for 7 d at 22°C under Bc light of 30 μmol m⁻² s⁻¹, Rc light of 50 μmol m⁻² s⁻¹, and FRc light of 10 μmol m⁻² s⁻¹. A to C, Images of seedlings. Bars = 1 cm. D, Bars represent means ± sd of hypocotyl lengths measured for 10 or more hypocotyls in each of three independent experiments.

Figure 6.

Photomorphogenesis and root elongation response of the wild type, phytochrome chromophore-deficient, and JA-insensitive mutants grown under Wc illumination with added MeJA. No-0 wild-type (WT), 35S::pBVR3, 35S::cBVR1, CAB3::pBVR2 (CAB-2), MERI5::pBVR1 (MERI-1), C24 wild-type, F3 seedlings of a M0062 × UASBVR cross (M0062/UASBVR), C20 wild-type, hy1-1, hy2-1, Col-0 wild-type, jar1, and myc2 lines were grown on Phytablend medium containing 1% (w/v) Suc with 20 μm MeJA for 10 d at 22°C under Wc illumination of 100 μmol m⁻² s⁻¹ A, Images of seedlings. Bars = 1 cm. B, Bars represent means ± sd of root lengths in mm (n ≥ 10 for each of six independent experiments). For statistical significance tests, comparisons were made relative to cognate wild-type lines: ^a P < 0.0001, ^b P < 0.01. For information on the range of seedling lengths observed for each line, see summarized frequency distribution data (Supplemental Table S1).

Figure 7.

Relative expression levels of JA-associated genes in wild-type, BVR-expressing, and mutant seedlings. Expression is shown for the JA biosynthetic gene OPR3 (A) and the JA-responsive marker gene VSP1 (B) in 10-d-old whole seedlings grown on Phytablend medium containing 1% (w/v) Suc with or without 20 μm MeJA at 22°C under Wc illumination of 100 μmol m⁻² s⁻¹. qPCR was conducted using RNA from No-0 wild-type (WT), 35S::pBVR3, 35S::cBVR1, CAB3::pBVR2, MERI5::pBVR1, C24 wild-type, M0062/UASBVR, C20 wild-type, hy1-1, hy2-1, Col-0 wild-type, jar1, and myc2 lines. Expression of UBC21 (At5g25760), which is a control gene encoding a ubiquitin-conjugating enzyme, was analyzed as a reference. Black bars, −JA; white bars, +JA (20 μm). Quantification by qPCR was performed with three independent experiments. Fold difference (Fold diff.) for levels of transcript accumulated in a test line relative to the cognate wild-type line is shown below each graph.