www.sciencesignaling.org/cgi/content/full/9/452/ra106/dc1 Supplementary Materials for Stem-piped light activates phytochrome B to trigger light responses in Arabidopsis thaliana roots Hyo-Jun Lee, Jun-Ho Ha, Sang-Gyu Kim, Han-Kyu Choi, Zee Hwan Kim, Yun-Jeong Han, Jeong-Il Kim, Youngjoo Oh, Variluska Fragoso, Kwangsoo Shin, Taeghwan Hyeon, Hong-Gu Choi, Kyung-Hwan Oh, Ian T. Baldwin,* Chung-Mo Park* *Corresponding author. Email: cmpark@snu.ac.kr (C.-M.P.); baldwin@ice.mpg.de (I.T.B.) Published 1 November 2016, Sci. Signal. 9, ra106 (2016) DOI: 10.1126/scisignal.aaf6530 The PDF file includes: Fig. S1. Schematic diagram of plant sample preparation for RNA sequencing. Fig. S2. Expression of photoreceptor genes in roots. Fig. S3. Plant growth for fluorescence imaging of phyb and HY5 distribution in root tissues. Fig. S4. Lateral root formation in grafted Col-0 and hy5-221 plants. Fig. S5. Primary root growth in soil-grown hy5-221 mutants. Fig. S6. Effects of growth hormones and sucrose on the nuclear import of phyb and HY5 stability in root cells. Fig. S7. Normalized light transmission through stem and stem-root segments. Fig. S8. Transmission of lights with different spectral compositions through stemroot segments. Fig. S9. Fluence rates and spectral compositions of stem-piped light. Fig. S10. Plant growth under soil light block conditions. Fig. S11. Experimental evaluation of the soil cover treatments. Fig. S12. Spectral composition of FR-rich light. Legends for tables S1 to S3 Table S4. Root genes that are differentially co-regulated by shoot light and root light. Table S5. Primers used in this study.
Other Supplementary Material for this manuscript includes the following: (available at www.sciencesignaling.org/cgi/content/full/9/452/ra106/dc1) Table S1 (Microsoft Excel format). Differentially expressed genes in the roots of plants having shoots in the dark and roots in the light. Table S2 (Microsoft Excel format). Differentially expressed genes in the roots of plants having shoots in the light and roots in the dark. Table S3 (Microsoft Excel format). Differentially expressed genes under both root-light and shoot-light conditions.
Fig. S1. Schematic diagram of plant sample preparation for RNA sequencing. Col-0 plants were grown at 23 o C under long days (LDs) on MS-phytagel media in plastic culture boxes. The culture boxes harboring two-week-old plants were incubated in complete darkness for 2 days to ensure complete phytochrome decay before exposing either the shoots or the roots to light for 1 day. The roots of the light-treated plants were harvested for total RNA extraction. Note that the MS-phytagel surface was covered with aluminum foil and fine soil particles, layered (3 mm in depth) onto the aluminum foil cover to block light transmission through the medium surface. L and D, light and dark, respectively. *The hole sizes were large enough to prevent direct contacts between growing seedlings and aluminum foil, which can be toxic to plants.
Fig. S2. Expression of photoreceptor genes in roots. (A) Transcript accumulation of photoreceptor genes. Col-0 plants were grown in soil at 23 o C under LDs until flowering. Plants were dissected into different organs for total RNA extraction. Transcript levels were examined by reverse transcription-mediated quantitative real-time PCR (qrt-pcr). Biological triplicates were averaged. Bars indicate standard error of the mean (SE). RL, rosette leaf; ST, stem; SA, shoot apex; FL, flower; R, root; CL, cauline leaf. (B) to (D) Histological detection of photoreceptor gene expression in root tissues. A -glucuronidase (GUS)-coding sequence was fused in-frame to the 3 ends of the promoter sequences of approximately 2 kbp upstream of the translational start site of PHYA (B), PHYB (C), and CRY2 (D), and the gene fusions were transformed into Col-0 plants. Transgenic plants were grown for 2 weeks on MS-agar plates before GUS staining. Arrows indicate protoxylem elements, and arrowheads indicate metaxylem elements.
Fig. S3. Plant growth for fluorescence imaging of phyb and HY5 distribution in root tissues. Two-week-old 35S:PHYB-GFP and 35S:HY5-GFP plants grown in soil were grown in the dark for 2 days. The shoots of the dark-grown plants were either left in the dark or exposed to light for 1 day. The root parts growing approximately 4 cm below the soil surface, as marked by arrows, were subjected to fluorescence imaging. We assume that the root parts used are not directly exposed to light through the soil layer under these growth conditions.
Fig. S4. Lateral root formation in grafted Col-0 and hy5-221 plants. Four-day-old plants grown on MS-agar plates were used for micrografting experiments. Grafted plants were incubated under LDs for 2 weeks on vertical MS-agar plates containing 0.5% sucrose. Lateral root density (lateral root number/cm) was calculated by dividing total lateral root number by primary root length. Fifteen measurements were averaged. Bars indicate SE. Note that lateral root density is not discernibly affected by the hy5 mutation.
Fig. S5. Primary root growth in soil-grown hy5-221 mutants. Plants were grown in soil for 2 weeks under LDs before measuring primary root lengths. Fifteen measurements were averaged. Bars indicate SE. Note that primary root growth is not affected by the hy5 mutation when grown in soil.
Fig. S6. Effects of growth hormones and sucrose on the nuclear import of phyb and HY5 stability in root cells. Three-week-old 35S:PHYB-GFP and 35S:HY5-GFP transgenic plants grown in soil were grown in complete darkness for 2 days. The dark-treated plants were transferred to liquid MS cultures containing either 100 M indole-3-acetic acid (IAA), 100 M methyl jasmonic acid (MeJA), or 100 mm sucrose and incubated in the dark for 1 day. For light and dark treatments, the dark-treated plants were transferred to liquid MS cultures and grown either in the light or in the dark for 1 day. GFP signals in root cells were visualized by differential interference contrast (DIC) and fluorescence microscopy. Scale bars, 50 m. Note that none of the tested chemicals influenced the subcellular distribution of phyb and the HY5 stability.
Fig. S7. Normalized light transmission through stem and stem-root segments. Stem and stem-root segments of ~3.5 cm were prepared from six-week-old Col-0 plants grown in soil for light transmission assays. (A) Spectrum of light source. Intensity of light source was measured in a range from 450 nm to 1000 nm for the normalization of transmitted light through stem and stem-root segments. a.u., arbitrary unit. (B and C) Normalized light transmission. Intensity of transmitted light through stem (B) and stem-root (C) segments was divided by the intensity of light source in the wavelength range examined in (A). Insets are amplified light spectra from 500 nm to 750 nm. Gray boxes indicate measurement saturation.
Fig. S8. Transmission of lights with different spectral compositions through stem-root segments. Light transmission assays were performed using stem-root segments of ~3.5 cm prepared from six-week-old Col-0 plants grown in soil. Fiber-coupled-warm white light source (Thorlabs, Cat No. MWWHF2), which emits light mostly in the green-to-red wavelength range (left panel), was applied into the upper end of stem-root segments. It is evident that the green-to-red light is efficiently transmitted through stem-root segments (right panel).
Fig. S9. Fluence rates and spectral compositions of stem-piped light. Stem-root segments with varying lengths (0.5 to 3.5 cm) were prepared from six-week-old Col-0 plants. Fluence rates were measured in a range from 450 nm to 1000 nm. Dotted lines denote 700 nm. Note that both fluence rates and spectral compositions of stem-piped light are affected by the different path lengths of light transmission. It is evident that the transmission of light with wavelengths of <700 nm is more rapidly reduced compared to that of light with longer wavelengths.
Fig. S10. Plant growth under soil light block conditions. Plants were grown in soil under LDs. After dark growth for 2 days, the plant pots harboring five-week-old plants were either exposed to light (left pot) or left in the dark (middle pot) for 1 day. For the soil-light block, the soil surface was covered with aluminum foil, and the foil was covered with fine soil particles (~3 cm in depth) to completely block light transmission through the soil layer and prevent direct contact between leaves and foil before exposing plants to light (right pot).
Fig. S11. Experimental evaluation of the soil cover treatments. (A) Experimental conditions. Three-week-old 35S:PHYB-GFP and 35S:HY5-GFP plants grown in soil were grown in the dark for 2 days. The shoot scions were removed, and the root stocks were either exposed to light (Light) or covered with either a 3-mm depth of fine soil particles (Soilcovered) or aluminum foil (Dark). (B and C) GFP signals in the root cells of 35S:PHYB-GFP and 35S:HY5-GFP plants (B and C, respectively) were visualized by DIC and fluorescence microscopy. (D) Fraction of cells exhibiting nuclear phyb. Four measurements, each with ~25 root cells, were averaged and statistically treated. Different letters represent a significant difference (P < 0.05) determined by one-way ANOVA with post hoc Tukey test. Bars indicate SE. (E) HY5 protein stability. Relative fluorescence intensities were averaged and statistically analyzed (n = 10). Different letters represent a significant difference (P < 0.05) determined by one-way ANOVA with post hoc Tukey test. Bars indicate SE.
Fig. S12. Spectral composition of FR-rich light. The spectral compositions of the FR-rich light source (Parus, Korea, Cat No. 111128-2) used in Figure 6 were measured.
Table S1. Differentially expressed genes in the roots of plants having shoots in the dark and roots in the light. This table is provided in a separate Excel file (aaf6530_table S1.xlsx). Two-week-old Col-0 plants grown on MS-phytagel media were grown in the dark for 2 days. The roots of the dark-grown plants were either exposed to light (root light) or left in the dark (whole dark) for 1 day before harvesting the roots for total RNA extraction and RNAsequencing. Expression levels of differentially expressed root genes exhibiting more than 4- fold changes with P value < 0.15 under root light conditions were compared to those under whole dark conditions. Biological triplicates were averaged. Table S2. Differentially expressed genes in the roots of plants having shoots in the light and roots in the dark. This table is provided in a separate Excel file (aaf6530_table S2.xlsx). Two-week-old Col-0 plants grown on MS-phytagel media were grown in the dark for 2 days. The shoots of the dark-grown plants were either exposed to light (shoot light) or left in the dark (whole dark) for 1 day before harvesting the roots for total RNA extraction and RNAsequencing. Expression levels of differentially expressed root genes exhibiting more than 4- fold changes with P value < 0.15 under shoot light conditions were compared to thosender whole dark conditions. Biological triplicates were averaged. Table S3. Differentially expressed genes under both root-light and shoot-light conditions. This table is provided in a separate Excel file (aaf6530_table S3.xlsx). Light-responsive root genes exhibiting more than 4-fold changes with P value < 0.15 under both shoot light and root light conditions were listed.
Gene locus Annotation Log2 value (D/L vs D/D) Log2 value (L/D vs D/D) AT1G01060 LHY, Late elongated hypocotyl 5.175 3.635 AT1G12370 PHR1, Photolyase 1* 12.538 10.552 AT1G20340 Plastocyanin 2 5.686 4.379 AT1G42970 GAPB, Glyceraldehyde-3-phosphate dehydrogenase B subunit 12.712 9.130 AT1G52560 HSP20-like chaperones superfamily protein 3.729 3.401 AT2G05100 LHCB2, Photosystem II light harvesting complex gene 2.1 12.843 11.456 AT2G29500 HSP20-like chaperones superfamily protein 2.291 2.454 AT2G37970 HBP2, SOUL heme-binding family protein 4.912 2.696 AT3G17609 HYH, HY5-homolog 7.667 6.339 AT3G51240 F3H, Flavanone 3-hydroxylase 8.743 3.303 AT4G32770 VTE1, Vitamin E deficient 1 8.275 5.508 AT4G38620 MYB4, Myb domain protein 4 3.764 3.305 AT5G04140 GLS1, Glutamate synthase 1 6.057 6.656 AT5G08020 RPA1B, RPA70-kDa subunit B 9.671 12.349 AT5G11260 HY5, Elongated hypocotyl 5* 3.751 2.562 AT5G13930 CHS, Chalcone synthase* 10.920 3.025 Table S4. Root genes that are differentially co-regulated by shoot light and root light. Light-responsive root genes analyzed by the Biological Networks Gene Ontology tool in Figure 1C were listed. Log2 values indicate fold changes. D/L indicates plants having the shoots in the dark and the roots in the light. L/D indicates plants having the shoots in the light and the roots in the dark. D/D indicates plants that were grown in the dark. Note that HY5 and its targets (PHR1 and CHS) are marked with asterisks.
Primers Sequences Usage eif4a-f 5 -TGACCACACAGTCTCTGCAA qrt-pcr eif4a-r 5 -ACCAGGGAGACTTGTTGGAC HY5-F 5 -TCGGAAAAGAAACTTCCGGT HY5-R 5 -TCCCTCGCTTCCTTTGACTT CHS-F 5 -ACATGCATCTGACGGAGGAA CHS-R 5 -GCTTAGGGACTTCGACCAC PHR1-F 5 -TTTGAAGGGAGCTTGGGAGT PHR1-R 5 -CATGAAACCGTGCATTTTCC PHYA-F 5 -ACAATGGCCAGTCATGCAGT PHYA-R 5 -ACATCTCCAAATCCAAGGGC PHYB-F 5 -TTGCATGCTAAAGGGTCCTG PHYB-R 5 -TAACCCGCTTGTTTGCAGTC CRY1-F 5 -AGCAGCGGAAGGAGAGAAAG CRY1-R 5 -TTTGTGAAAGCCGTCTCCAG CRY2-F 5 -GGTCTCCAGGATGGAGCAAT CRY2-R 5 -GGCACACTGGAAAACGTGTC PHOT1-F 5 -ACTGAAGGGGCCAAAGAAAA PHOT1-R 5 -TTCATCGCTCTCCGATTTTG PHOT2-F 5 -GCCAGACACCGACAAGAATG PHOT2-R 5 -CCTGCATCCCAATGAACTTG Table S5. Primers used in this study. The primers used were designed using the Primer3 software (version 0.4.0, http://primer3.sourceforge.net/releases.php) to ensure that they have calculated melting temperatures in the range of 50 60 o C. F, forward primer; R, reverse primer.