SUPPLEMENTARY INFORMATION - Nature Research€¦ · SUPPLEMENTAR ORMATION DOI: 10.1038/NPLANTS.2016.13 JUB1 ATEXP8 ATEXP1 ATEXP19 ATEXP11 ATEXP10 ATEXP3 ATEXP4 ATEXP6 ATEXP13 ATEXP12

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NPLANTS.2016.13

NATURE PLANTS | www.nature.com/natureplants 1

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Supplementary Figure 1

Supplementary Figure 1. Enhanced hypocotyl elongation and cell size in JUB1-amiRNA plants. (a) Hypocotyl length of JUB1-amiRNA compared to WT seedlings grown for 7 days in the dark. Means ± SD (n = 20) and **P 0.01 by Student’s t-test. (b) Epidermal cell size in JUB1-amiRNA and WT measured at three different positions in the middle of the leaf blade. Measurements were taken for leaf no. 5 of 4-week-old soil-grown plants. Means ± SE (n = 5) and **P < 0.01 by Student’s t-test. Asterisks in graphs denote significant differences relative to WT.

http://dx.doi.org/10.1038/nplants.2016.13

2 NATURE PLANTS | www.nature.com/natureplants

SUPPLEMENTARY INFORMATION DOI: 10.1038/NPLANTS.2016.13

WT JUB1-OX

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Supplementary Figure 2. JUB1 negatively regulates cell elongation. (a) Microscope image of adaxial epidermal cells of fully expanded 5th leaf of 4-week-old rosettes and quantification data (lower panel). Cell size was measured at three different positions in the middle of the leaf blade. Error bars represent SE (n = 5), **P < 0.01 by Student’s t-test. (b) Heat map of the EXPANSIN genes differentially expressed between JUB1 transgenics and WT. The log2 fold change scale is indicated at the left side of the heat map (see Supplementary Table 1 for corresponding data).





ATG

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ATG DWF4

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ATG RGL1

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BZR1 -738

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Supplementary Figure 3. Schematic representation of the JUB1 binding site in the promoters of genes differentially regulated by JUB1. A diagram of GA3ox1, DWF4, GAI, RGL1, PIF4 and BZR1 promoters showing the position of JUB1 binding sites and ChIP amplicons (black thick underlines).



SUPPLEMENTARY INFORMATION DOI: 10.1038/NPLANTS.2016.13Supplementary Figure 4

GA3ox1prom:GUS

DWF4prom:GUS

h

JUB1-OX / GA3ox1prom:GUS

JUB1-OX / DWF4prom:GUS

Supplementary Figure 4. Reduction of GA3ox1prom:GUS and DWF4prom:GUS activity upon accumulation of JUB1. Histochemical GUS staining of transgenic lines harbouring 3-kb and 1,132-bp promoter fragments of GA3ox1 (GA3ox1prom:GUS) and DWF4 (DWF4prom:GUS), respectively, in Col-0 (left panels) and JUB1-OX backgrounds (right panels) in various tissues at different stages of plant development. Tissues from top to bottom; 5-day-old seedling, rosette of 14-day-old seedling, developing flower, 3-day-old light-grown seedling, pedicel, and developing flower. In JUB1-OX/GA3ox1prom:GUS seedlings the expression of GA3ox1 is strongly decreased in the stem and is restricted to the vasculature near the leaf tips. In flowers, GA3ox1 expression is only detected at very low level at the base of flowers and mostly fades in the stamen filaments. In JUB1-OX/DWF4prom:GUS lines expression of DWF4 is reduced in tissues joining roots and hypocotyls, and absent in root tips (higher magnification of tissues is indicated by arrows). A decrease of DWF4 expression upon JUB1 overexpression is also observed in developing pedicels. In flowers, JUB1 overexpression reduces DWF4 expression in anthers and in filaments and the base of flower buds.




BRZ

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Supplementary Figure 5. Altered response of JUB1 transgenic plants to GA, BR, PAC and BRZ treatments provide evidence for the role of JUB1 in GA and BR biosynthesis. (a) JUB1-OX seedlings show insensitivity to treatments with PAC and BRZ. Hypocotyl length of 7-day-old JUB1 transgenic and WT seedlings grown on MS medium supplemented with mock solution (mock), 1 μM PAC or 1 μM BRZ. Asterisks denote significant differences relative to mock-treated seedlings. (b) Effect of PAC and BRZ on light-grown JUB1 transgenic and WT seedlings grown on MS medium either with 1 μM BRZ or 2 μM PAC for 2 weeks. (c) Application of GA4 (0.2 μM) or BL (1 μM) partially restores the short-hypocotyl phenotype of 7-day-old JUB1-OX seedlings in the dark. The full recovery of phenotype is observed only after combined application of GA and BR. Asterisks denote significant differences relative to WT. (d) Effect of GA4 and GA9 on vegetative (upper panel) and reproductive (lower panel) growth of JUB1-OX seedlings. Photographs were taken 3 days (upper panel) and 10 days (lower panel) after transfer of 2-week-old seedlings to the GA-containing media. Arrows indicate early-induced flowering in response to GA4 treatment. Error bars in a and c indicate SD (n = 20) and **P 0.01 by Student’s t-test and NS = not significant. (e) Effect of GA4 and GA9 on jub1-1 and WT seedlings. Photographs were taken three days after the transfer to the GA-containing media. Arrows indicate early induced flowering in response to hormone treatment.




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WT bzr1-1D JUB1-OX/ bzr1-1D

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Supplementary Figure 6. Overexpression of JUB1 in BR signal-enhanced mutant, bzr1-1D, significantly recovers JUB1-OX phenotypes. (a) Expression level of JUB1, in bzr1-1D and different lines of JUB1-OX/bzr1-1D (numbered 1, 2 and 5) quantified by qRT-PCR. Means ± SD (n = 3). (b) Short hypocotyl length of JUB1-OX seedlings is restored in the bzr1-1D mutant background. Seedlings were grown in the dark for 7 days. Asterisks denote significant differences relative to WT. (c) Phenotype of developing flowers in WT, bzr1-1D, JUB1-OX/bzr1-1D and JUB1-OX plants. (d) Recovery of the small-silique phenotype of JUB1-OX plants in the bzr1-1D mutant background. The silique length was measured for four siliques located in the middle of the flower stalk. Asterisks denote significant differences relative to JUB1-OX. Error bars in b and d are means ± SD of 20 biological replicates. **P < 0.01 by Student’s t-test and NS = not significant.




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typhasterol (TY)

castasterone (CS)

brassinolide (BL)

teasterone (TE)

cathasterone (CT)


Supplementary Figure 7. JUB1 alters the levels of bioactive GAs and BRs. (a) Simplified GA biosynthesis pathway and concentration of GA isoforms in 10-day-old jub1-1, WT and 35S:JUB1-GFP seedlings. Means (pg/mg fresh weight) ± SD (n = 5). (b) Simplified BR biosynthesis pathway and BR profile analyses of 4-week-old soil-grown jub1-1, WT and 35S:JUB1-GFP plants. Means (pg/mg fresh weight) ± SD (n = 3). In a and b *P < 0.05, **P < 0.01 by Student’s t-test and NS = not significant. Asterisks denote significant differences relative to WT.




JUB1 ATEXP8 ATEXP1 ATEXP19 ATEXP11 ATEXP10 ATEXP3 ATEXP4 ATEXP6 ATEXP13 ATEXP12 ATEXP5 ATEXLA3 ATEXPR ATEXPB3 ATEXP2 ATEXP14

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Supplementary Figure 8. Reduced cell size of JUB1-OX plants is fully rescued upon overexpression of GA3ox1 and DWF4. (a) Comparison of adaxial epidermal cell sizes in rosette leaves of Triple-OX and WT leaves. Measurements were taken for leaf no. 5 of 4-week-old soil-grown plants. Cell size was measured at three different positions in the middle of the leaf blade. Means ± SD (n = 3), **P < 0.01 by Student’s t-test. Asterisks denote significant difference relative to WT. (b) Heat map showing transcript abundance of EXPANSIN genes differentially expressed between jub1-1 or JUB1-OX and WT plants (presented in Supplementary Fig. 2b) in Triple-OX seedlings compared to WT. The log2 fold change scale is indicated at the left side of the heat map (see Supplementary Table 1 for corresponding data).




JUB1-OX WT JUB1/ DWF4-OX

JUB1/ GA3ox1-OX

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Supplementary Figure 9. Leaf shape, delayed senescence and stress tolerance of JUB1-OX plants are suppressed upon overexpression of GA3ox1 and DWF4. (a) Leaf shape and senescence phenotype; leaf number 9 detached from plants (45 DAS). (b) Chlorophyll content of the five biggest leaves of transgenic and WT plants at 45 DAS. SPAD values were determined at three different positions in the middle of each leaf. Means ± SD (n = 10), **P < 0.01 by Student’s t-test. (c) Percentage of survived leaves which had at least ~50% green blades (inspected visually) at 45 DAS. Means ± SD (n = 10), *P < 0.05 and **P < 0.01 by Student’s t-test. Asterisks in graphs b and c denote significant differences relative to JUB1-OX. (d) Expression of the senescence marker gene SAG12 in transgenic plants compared to WT. FC, fold change. Means ± SD (n = 3). (e) Phenotype of JUB1 transgenics and WT seedlings upon NaCl treatment. Two-week-old seedlings were transferred to media containing 150 mM NaCl. Photographs were taken two days later. (f) Phenotype of JUB1 transgenics and WT seedlings upon heat treatment. Ten-day-old seedlings were transferred to 45°C for 45 min. Photographs were taken eight days later. (g) Expression of DREB2A measured by qRT-PCR in transgenic plants compared to WT 35 DAS. FC, fold change. Means ± SD (n = 3).




penta della JUB1-OX/ penta della

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Supplementary Figure 10. Stress tolerance of JUB1-OX plants is suppressed in JUB1-OX/penta della plants. (a) Expression level of JUB1, GA3ox1 and DWF4 in JUB1-OX/Ler and JUB1-OX/penta della lines, quantified by qRT-PCR. Means ± SD (n = 3). (b) Phenotype of transgenic lines and Ler upon heat treatment. Nine-day-old seedlings were transferred to 45°C for 45 min. Photographs were taken 4 days later. (c) Phenotype of transgenic lines and Ler upon NaCl treatment. Two-week-old seedlings were transferred to media containing 150 mM NaCl. Photographs were taken 2 days after the transfer to the NaCl-containing media.




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Supplementary Figure 11. PIF4 and BZR1 bind to the promoter of JUB1 and repress its transcription. (a) Binding of PIF4 and BZR1 to the promoter of JUB1 in Y1H assay. The JUB1 promoter with the position of the G-box element (highlighted in red), the putative binding site of PIF4 and BZR1, is shown in the lower part of the panel. (b) Transient expression assay in N. benthamiana leaves using JUB1prom:GUS as a reporter. GUS mRNA levels were analysed by RT-qPCR. Means ± SD (n = 3). Empty plasmids (-) were used as negative controls. (c) Expression level of GA3ox1 and DWF4 in PIF4 and BZR1 transgenic lines, quantified by qRT-PCR. Means ± SD (n = 3). (d, e) Hypocotyl length of 7-day-old seedlings grown in darkness. Asterisks denote significant differences relative to (d) PIF4-OX or (e) 35S:BZR1-HA. (f) Partial recovery of flower defects and (g) the small-silique phenotype of JUB1-OX plants in the 35S:BZR1-HA background. Silique length was measured for four siliques located in the middle of the flower stalk. Asterisks denote significant differences relative to JUB1-OX. Error bars in d, e and g are means ± SD of 20 biological replicates. **P < 0.01 by Student’s t-test.




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Supplementary Figure 12. Enhanced expression of JUB1 upon GA and BR treatments. JUB1 expression in WT plants treated with 1 μM GA3, 1 μM GA4, 10 nM BL or a combination of these hormones compared to mock treatment (no hormones added). Means ± SD (n = 3) and FC, fold change.


0123456789

pDEST32 (Bait)

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Supplementary Figure 13. JUB1 physically interacts with DELLAs. Yeast-two-hybrid (Y2H) assays analysing the pairwise interaction of JUB1 with three DELLA proteins (GAI, RGA, and RGL1). Relative b-galactosidase (b-Gal) activity was measured in liquid assays using chlorophenol red-b-galactopyranoside (CPRG) as substrate. Empty bait or prey plasmids (-) were used as negative controls. Means ± SD of b-Gal activity (1 unit = 10-4 min-1 mL-1) were obtained from at least six independent colonies.




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Supplementary Figure 12. Enhanced expression of JUB1 upon GA and BR treatments. JUB1 expression in WT plants treated with 1 μM GA3, 1 μM GA4, 10 nM BL or a combination of these hormones compared to mock treatment (no hormones added). Means ± SD (n = 3) and FC, fold change.


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Supplementary Figure 13. JUB1 physically interacts with DELLAs. Yeast-two-hybrid (Y2H) assays analysing the pairwise interaction of JUB1 with three DELLA proteins (GAI, RGA, and RGL1). Relative b-galactosidase (b-Gal) activity was measured in liquid assays using chlorophenol red-b-galactopyranoside (CPRG) as substrate. Empty bait or prey plasmids (-) were used as negative controls. Means ± SD of b-Gal activity (1 unit = 10-4 min-1 mL-1) were obtained from at least six independent colonies.




1

Supplementary Table 1. Transcript ratio (log2 fold change) of EXPANSIN genes differentially expressed between 2-week-old JUB1 transgenic and WT plants. All values are the mean of three independent experiments ± SD. Asterisks indicate significant differences between JUB1 transgenic and WT plants. (*P < 0.05, Students t-test).

AGI Gene name JUB1-OX/WT

JUB1- amiRNA/WT Triple-OX/WT

AT2g43000 JUB1 5.71±0.5* -3.27±0.4* 4.24±0.4* AT2G40610 ATEXP8 -2.76±0.2* 0.91±0.2* 1.32±0.3* AT1G69530 ATEXP1 -2.83±0.3* 1.22±0.3* 1.11±0.2* AT3G29365 ATEXP19 -1.79±0.2* 0.89±0.1* 1.45±0.2* AT1G20190 ATEXP11 -3.37±0.3* 0.76±0.1* 0.77±0.2* AT1G26770 ATEXP10 -1.89±0.2* 0.63±0.0* 0.52±0.2 AT2G37640 ATEXP3 -1.38±0.1* 0.20±0.2 0.73±0.1* AT2G39700 ATEXP4 -7.91±0.4* 0.80±0.1* 0.92±0.2* AT2G28950 ATEXP6 -0.84±0.1* -0.03±0.3 0.26±0.2 AT3G03220 ATEXP13 -0.28±0.2 0.12±0.3 0.54±0.3 AT3G15370 ATEXP12 -4.54±0.4* 0.14±0.2 0.45±0.2 AT3G29030 ATEXP5 -3.45±0.5* 0.44±0.3 0.59±0.2 AT3G45960 ATEXLA3 -3.01±0.2* 0.26±0.2 0.35±0.3 AT4G17030 ATEXPR -2.98±0.2* 0.05±0.2 0.45±0.3 AT4G28250 ATEXPB3 -6.77±0.4* -0.02±0.2 0.37±0.3 AT5G05290 ATEXP2 -2.22±0.4* 0.03±0.3 0.55±0.3 AT5G56320 ATEXP14 -2.33±0.4* 0.29±0.2 0.51±0.3

2

Supplementary Table 2. Flowering time of JUB1 transgenic and WT plants. Number of rosette leaves was recorded on bolting plants. Each value is the mean ± SD of three replications (12 plants were analysed in each replication). Flowering/bolting time was determined by counting the days from sowing to the first elongation of the floral stem at 1 mm height.

Line Days to flower

Number of rosette leaves

jub1-1 24.4±0.5 13.6±0.3 JUB1-amiRNA 24.5±0.5 13.5±0.2 WT 26.4±0.4 13.2±0.1 Triple-OX 26.5±0.5 13.5±0.3 JUB1/DWF4-OX 28.6±0.6 14.2±0.3 JUB1/GA3ox1-OX 29.8±0.5 16.8±0.2 JUB1-OX 33.2±0.4 18.2±0.1




1

Supplementary Table 1. Transcript ratio (log2 fold change) of EXPANSIN genes differentially expressed between 2-week-old JUB1 transgenic and WT plants. All values are the mean of three independent experiments ± SD. Asterisks indicate significant differences between JUB1 transgenic and WT plants. (*P < 0.05, Students t-test).

AGI Gene name JUB1-OX/WT

JUB1- amiRNA/WT Triple-OX/WT

AT2g43000 JUB1 5.71±0.5* -3.27±0.4* 4.24±0.4* AT2G40610 ATEXP8 -2.76±0.2* 0.91±0.2* 1.32±0.3* AT1G69530 ATEXP1 -2.83±0.3* 1.22±0.3* 1.11±0.2* AT3G29365 ATEXP19 -1.79±0.2* 0.89±0.1* 1.45±0.2* AT1G20190 ATEXP11 -3.37±0.3* 0.76±0.1* 0.77±0.2* AT1G26770 ATEXP10 -1.89±0.2* 0.63±0.0* 0.52±0.2 AT2G37640 ATEXP3 -1.38±0.1* 0.20±0.2 0.73±0.1* AT2G39700 ATEXP4 -7.91±0.4* 0.80±0.1* 0.92±0.2* AT2G28950 ATEXP6 -0.84±0.1* -0.03±0.3 0.26±0.2 AT3G03220 ATEXP13 -0.28±0.2 0.12±0.3 0.54±0.3 AT3G15370 ATEXP12 -4.54±0.4* 0.14±0.2 0.45±0.2 AT3G29030 ATEXP5 -3.45±0.5* 0.44±0.3 0.59±0.2 AT3G45960 ATEXLA3 -3.01±0.2* 0.26±0.2 0.35±0.3 AT4G17030 ATEXPR -2.98±0.2* 0.05±0.2 0.45±0.3 AT4G28250 ATEXPB3 -6.77±0.4* -0.02±0.2 0.37±0.3 AT5G05290 ATEXP2 -2.22±0.4* 0.03±0.3 0.55±0.3 AT5G56320 ATEXP14 -2.33±0.4* 0.29±0.2 0.51±0.3

2

Supplementary Table 2. Flowering time of JUB1 transgenic and WT plants. Number of rosette leaves was recorded on bolting plants. Each value is the mean ± SD of three replications (12 plants were analysed in each replication). Flowering/bolting time was determined by counting the days from sowing to the first elongation of the floral stem at 1 mm height.

Line Days to flower

Number of rosette leaves

jub1-1 24.4±0.5 13.6±0.3 JUB1-amiRNA 24.5±0.5 13.5±0.2 WT 26.4±0.4 13.2±0.1 Triple-OX 26.5±0.5 13.5±0.3 JUB1/DWF4-OX 28.6±0.6 14.2±0.3 JUB1/GA3ox1-OX 29.8±0.5 16.8±0.2 JUB1-OX 33.2±0.4 18.2±0.1




3

Supplementary Table 3. Transcript ratio (log2 fold change) of GA and BR biosynthesis/signalling genes differentially expressed between JUB1 transgenic and WT plants. Data were obtained for whole shoots at 14 and 35 DAS, from three biological replications. Asterisks indicate significant differences between JUB1 transgenic and WT plants. (*P < 0.05, Students t-test).

AGI

Gene/ protein name

JUB1-OX/WT JUB1-amiRNA/ WT 14 DAS

35 DAS

14 DAS 35 DAS

AT2g43000 JUB1 5.58±0.4* 8.15±1.1* -3.06±0.3* -5.64±0.6*

AT1G15550 GA3ox1 -7.52±0.7* -2.1±0.1* 0.96±0.2* 1.22±0.1*

AT1G80340 GA3ox2 -0.85±0.2* -0.9±0.2* 0.43±0.2 0.69±0.2*

AT1G14920 GAI 1.11±0.3* 0.71±0.1* -0.2±0.2 -0.1±0.3

AT1G66350 RGL1 2.44±0.3* 1.03±0.3* 0.11±0.3 0.08±0.3

AT2G43010 PIF4 -1.35±0.2* -0.68±0.1* 0.18±0.3 0.104±0.3

AT3G50660 DWF4 -5.31±0.4* -1.60±0.3* 1.2±0.2* 0.90±0.2*

AT5G38970 BR6ox1 -5.86±0.6* -0.96±0.2* 0.49±0.3 1.50±0.3*

AT4G30610 BRS1 -5.23±0.3* -0.49±0.3 0.54±0.3 2.08±0.3*

AT5G57560 TCH4 -2.09±0.2* -2.50±0.4* 0.00±0.2 0.40±0.3

AT1G18400 BEE1 -5.7±0.4* -1.00±0.2* 0.83±0.2* 0.10±0.2

AT2G43060 IBH1 1.46±0.2* 0.59±0.3 0.09±0.3 -0.97±0.2*

AT1G75080 BZR1 -0.96±0.1* -0.43±0.2 0.13±0.3 0.02±0.2


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SUPPLEMENTARY INFORMATION - Nature Research€¦ · SUPPLEMENTAR ORMATION DOI: 10.1038/NPLANTS.2016.13 JUB1 ATEXP8 ATEXP1 ATEXP19 ATEXP11 ATEXP10 ATEXP3 ATEXP4 ATEXP6 ATEXP13 ATEXP12