universal stress protein hru1 mediates the universal ... · the universal stress protein hru1...

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NPLANTS.2015.151

NATURE PLANTS | www.nature.com/natureplants 1

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The Universal Stress Protein HRU1 mediates ROS homeostasis under anoxia

Silvia Gonzali, Elena Loreti, Francesco Cardarelli, Giacomo Novi, Sandro Parlanti, Chiara

Pucciariello, Laura Bassolino, Valeria Banti, Francesco Licausi, Pierdomenico Perata

Universal stress protein HRU1 mediates ROS homeostasis under anoxia

http://dx.doi.org/10.1038/nplants.2015.151

2 NATURE PLANTS | www.nature.com/natureplants

SUPPLEMENTARY INFORMATION DOI: 10.1038/NPLANTS.2015.151

2

Supplementary Methods

Plant materials and growth conditions. Genotypes used included Arabidopsis thaliana, Col-0 ecotype, T-DNA

insertion mutants hru1-1 (SALK_042828)51, hru1-2 (SALK_136373)51 and rhd2-1 (NASC N2259). Mutant lines were

obtained from the European Arabidopsis Stock Centre (NASC) and homozygotes were established. In hru1-2, a T-DNA

insertion 98 nucleotides upstream ATG in the promoter region of AT3G03270 resulted in no expression of the gene

(Supplementary Fig. 2). Double mutant lines were obtained by crossing the hru1-1 and rhd2-1 mutants. To complement

the hru1-1 mutation, hru1-1 was crossed with the 35S:HRU1 line. For experiments with 4-day-old seedlings, sterilized

seeds were sown in liquid Murashige–Skoog half-strength medium supplemented with 1% (w/v) sucrose. Seeds were

stratified for 72 h in the dark at 4°C and then transferred at 23°C with a 12-h light (100 mE m2 sec2 intensity) photoperiod

with shaking. To obtain plants grown on vertical agar plates, seeds were germinated on agar (0.9% w/v) medium

supplemented with 1% (w/v) sucrose using the same conditions as reported above. Anoxia treatment assays were

carried out in dim light starting at 8:00 a.m.45. An enclosed anaerobic workstation (Anaerobic System model 1025;

Forma Scientific) was used to provide an oxygen-free environment for seedling incubation. This chamber uses

palladium catalyst wafers and desiccant wafers to maintain strict anaerobiosis to less than 10 μg mL–1 oxygen

according to the manufacturer’s specifications). High-purity N2 was used to initially purge the chamber, and the

working anaerobic gas mixture was N2 :H2 with a ratio of 90:10.

Gene cloning. Arabidopsis genomic DNA was extracted from a single leaf using the “Wizard® Genomic DNA

Purification Kit” (Promega, Madison, WI, US). AT3G03270 genomic sequence was amplified by PCR starting from 20

ng genomic DNA using the “Phusion® High-Fidelity DNA Polymerase” and the primers listed in Supplementary Table

2. 0.5 μg RNA extracted from Arabidopsis seedlings subjected to a 4 h anoxia treatment were reverse transcribed using

the SuperScript® III Reverse Transcriptase (Life Technologies, CA, US) according to the manufacturer’s protocol.

Starting from the resulting cDNA, the CDS sequences of HRU1 (At3g03270.2), HRU1tr (corresponding to the CDS

sequence of AT3G03270 upstream the T-DNA insertion in the mutant line SALK_042828), ROP2 (AT1G20090),

RbohD (AT5G47910), and TRXh (AT3G51030) were amplified by PCR using the Phusion® High-Fidelity DNA

Polymerase (New England BioLabs Inc., MA, US) and the primers listed in Supplementary Table 2. For ROP2 and

RbohD, the CDS was cloned with and without the stop codon, enabling the corresponding ORF to be joined in frame

with a N-terminal or C-terminal tag, respectively. For ROP2 cloning, forward and reverse primers were designed on

5’UTR and 3’UTR, respectively, due to the high degree of similarity of the CDS with other ROP encoding genes. For

CA-ROP2 and DN-ROP2 cloning, see the section on Y2H. Each amplified sequence was then cloned into pENTR/D-

TOPO® vector (Life Technologies). The entry clones were recombined with different destination vectors, as described

in the following subsections, via “Gateway® Recombination Cloning Technology” (Life Technologies).

Constructs and transgenic plant preparation. The genomic sequence of AT3G03270 subcloned into pENTR/D-

TOPO as described above was recombined with the destination vector pK7WG252 to obtain the expression vector

carrying the construct 35S:HRU1. Transgenic plants were obtained using the floral dip method53. T0 seeds were

3

screened for kanamycin resistance, and transgenic lines were identified by PCR on genomic DNA (Supplementary

Table 3).

Protein localizations and Bimolecular Fluorescence Complementation assay. To obtain the GFP tagged expression

of HRU1, ROP2 and RbohD, the corresponding entry vectors, obtained as described above, were recombined with

p2FGW7 plasmids52. For BiFC assays, the Gateway® compatible destination vectors used were pDH51-GW-YFPN and

pDH51-GW-YFPC, enabling fusion of the N-terminus or C-terminus of the yellow fluorescent protein (YFP) moieties,

respectively, to the C-terminus of the protein of interest54 (http://arabidopsis.info). Control vectors were pDH51-YFPC

and pDH51-YFPN. The vectors pE-SPYNE-GW and pE-SPYCE-GW, enabling fusion of the N-terminus or C-terminus

of YFP moieties, respectively, to the N-terminus of the protein of interest (based on the vectors described55,56) were also

used. Control vectors were pE-SPYCE(-) and pE-SPYNE(-). Arabidopsis mesophyll protoplasts were prepared46 and

transformed with plasmid DNA. For protein localization, 10 µg assays of plasmid DNA were used for protoplast

transformation. As a positive control for transfection, the pAVA393 construct57, was used encoding a cytosolic GFP

protein. For BiFC assays the amount of plasmid DNA used in each transformation was the lowest that resulted in a

positive interaction between the proteins tested and a negative signal in the control experiments (Supplementary Fig.

7). After transformation, protoplasts were incubated in the dark at 25°C for 16 h before subsequent fluorescence

analysis.

Split-luciferase. Gateway® compatible destination vectors used for split-luciferase assay were from the pDuEx

series58. The entry clone of HRU1 (AT3G03270.2) or HRU1tr, obtained as described above, was recombined with

pDuExAN6 (leading to the production of the chimeric proteins NRLuc-HRU1 and NRLuc-HRU1tr, respectively) and

the entry vector of TRXh was recombined with pDuExDN6 (chimeric protein CRLuc-TRXh). Five μg of each of the

two recombined vectors harboring the two halves of the Renilla Luciferase fused with the genes of interest were co-

transfected in Arabidopsis mesophyll protoplasts46. Single vectors were transfected as negative controls. The samples

were then incubated in the dark at 25°C for 16 hr before subsequent luminescence analysis. This was carried out using

the “Renilla Luciferase Assay System” (Promega). Protoplasts were precipitated by centrifugation (1,000 x g for 2 min)

and resuspended in 30 μl of Renilla Luciferase Assay Lysis Buffer. Then 6 μl of each cell lysate were added to 30 μl of

Renilla Luciferase Assay Reagent and luminescence was immediately measured with a Lumat LB 9507 Tube

Luminometer (Berthold Technologies, NY, USA). For luminescence quantification, five biological replicates were

analyzed for each protein-protein interaction.

Total RNA extraction, qPCR and RNA gel blots. RNA extraction, removal of genomic DNA, cDNA synthesis and

qRT-PCR analyses were performed as described previously47with a minor modification (omission of aurintricarboxylic

acid) to make the protocol compatible with the subsequent PCR procedures. GAPDH and 40S rRNA were used as

reference genes. Three replicates for each experiment were used and the average expression value was calculated. For a

list of the primers used and designed using QuantPrime (http://quantprime.mpimp-golm.mpg.de/)48 see Supplementary

Table 4. RNA gel blots were performed as previously described42 using probes produced using the primers reported in

Supplementary Table 4.




2

Supplementary Methods

Plant materials and growth conditions. Genotypes used included Arabidopsis thaliana, Col-0 ecotype, T-DNA

insertion mutants hru1-1 (SALK_042828)51, hru1-2 (SALK_136373)51 and rhd2-1 (NASC N2259). Mutant lines were

obtained from the European Arabidopsis Stock Centre (NASC) and homozygotes were established. In hru1-2, a T-DNA

insertion 98 nucleotides upstream ATG in the promoter region of AT3G03270 resulted in no expression of the gene

(Supplementary Fig. 2). Double mutant lines were obtained by crossing the hru1-1 and rhd2-1 mutants. To complement

the hru1-1 mutation, hru1-1 was crossed with the 35S:HRU1 line. For experiments with 4-day-old seedlings, sterilized

seeds were sown in liquid Murashige–Skoog half-strength medium supplemented with 1% (w/v) sucrose. Seeds were

stratified for 72 h in the dark at 4°C and then transferred at 23°C with a 12-h light (100 mE m2 sec2 intensity) photoperiod

with shaking. To obtain plants grown on vertical agar plates, seeds were germinated on agar (0.9% w/v) medium

supplemented with 1% (w/v) sucrose using the same conditions as reported above. Anoxia treatment assays were

carried out in dim light starting at 8:00 a.m.45. An enclosed anaerobic workstation (Anaerobic System model 1025;

Forma Scientific) was used to provide an oxygen-free environment for seedling incubation. This chamber uses

palladium catalyst wafers and desiccant wafers to maintain strict anaerobiosis to less than 10 μg mL–1 oxygen

according to the manufacturer’s specifications). High-purity N2 was used to initially purge the chamber, and the

working anaerobic gas mixture was N2 :H2 with a ratio of 90:10.

Gene cloning. Arabidopsis genomic DNA was extracted from a single leaf using the “Wizard® Genomic DNA

Purification Kit” (Promega, Madison, WI, US). AT3G03270 genomic sequence was amplified by PCR starting from 20

ng genomic DNA using the “Phusion® High-Fidelity DNA Polymerase” and the primers listed in Supplementary Table

2. 0.5 μg RNA extracted from Arabidopsis seedlings subjected to a 4 h anoxia treatment were reverse transcribed using

the SuperScript® III Reverse Transcriptase (Life Technologies, CA, US) according to the manufacturer’s protocol.

Starting from the resulting cDNA, the CDS sequences of HRU1 (At3g03270.2), HRU1tr (corresponding to the CDS

sequence of AT3G03270 upstream the T-DNA insertion in the mutant line SALK_042828), ROP2 (AT1G20090),

RbohD (AT5G47910), and TRXh (AT3G51030) were amplified by PCR using the Phusion® High-Fidelity DNA

Polymerase (New England BioLabs Inc., MA, US) and the primers listed in Supplementary Table 2. For ROP2 and

RbohD, the CDS was cloned with and without the stop codon, enabling the corresponding ORF to be joined in frame

with a N-terminal or C-terminal tag, respectively. For ROP2 cloning, forward and reverse primers were designed on

5’UTR and 3’UTR, respectively, due to the high degree of similarity of the CDS with other ROP encoding genes. For

CA-ROP2 and DN-ROP2 cloning, see the section on Y2H. Each amplified sequence was then cloned into pENTR/D-

TOPO® vector (Life Technologies). The entry clones were recombined with different destination vectors, as described

in the following subsections, via “Gateway® Recombination Cloning Technology” (Life Technologies).

Constructs and transgenic plant preparation. The genomic sequence of AT3G03270 subcloned into pENTR/D-

TOPO as described above was recombined with the destination vector pK7WG252 to obtain the expression vector

carrying the construct 35S:HRU1. Transgenic plants were obtained using the floral dip method53. T0 seeds were

3

screened for kanamycin resistance, and transgenic lines were identified by PCR on genomic DNA (Supplementary

Table 3).

Protein localizations and Bimolecular Fluorescence Complementation assay. To obtain the GFP tagged expression

of HRU1, ROP2 and RbohD, the corresponding entry vectors, obtained as described above, were recombined with

p2FGW7 plasmids52. For BiFC assays, the Gateway® compatible destination vectors used were pDH51-GW-YFPN and

pDH51-GW-YFPC, enabling fusion of the N-terminus or C-terminus of the yellow fluorescent protein (YFP) moieties,

respectively, to the C-terminus of the protein of interest54 (http://arabidopsis.info). Control vectors were pDH51-YFPC

and pDH51-YFPN. The vectors pE-SPYNE-GW and pE-SPYCE-GW, enabling fusion of the N-terminus or C-terminus

of YFP moieties, respectively, to the N-terminus of the protein of interest (based on the vectors described55,56) were also

used. Control vectors were pE-SPYCE(-) and pE-SPYNE(-). Arabidopsis mesophyll protoplasts were prepared46 and

transformed with plasmid DNA. For protein localization, 10 µg assays of plasmid DNA were used for protoplast

transformation. As a positive control for transfection, the pAVA393 construct57, was used encoding a cytosolic GFP

protein. For BiFC assays the amount of plasmid DNA used in each transformation was the lowest that resulted in a

positive interaction between the proteins tested and a negative signal in the control experiments (Supplementary Fig.

7). After transformation, protoplasts were incubated in the dark at 25°C for 16 h before subsequent fluorescence

analysis.

Split-luciferase. Gateway® compatible destination vectors used for split-luciferase assay were from the pDuEx

series58. The entry clone of HRU1 (AT3G03270.2) or HRU1tr, obtained as described above, was recombined with

pDuExAN6 (leading to the production of the chimeric proteins NRLuc-HRU1 and NRLuc-HRU1tr, respectively) and

the entry vector of TRXh was recombined with pDuExDN6 (chimeric protein CRLuc-TRXh). Five μg of each of the

two recombined vectors harboring the two halves of the Renilla Luciferase fused with the genes of interest were co-

transfected in Arabidopsis mesophyll protoplasts46. Single vectors were transfected as negative controls. The samples

were then incubated in the dark at 25°C for 16 hr before subsequent luminescence analysis. This was carried out using

the “Renilla Luciferase Assay System” (Promega). Protoplasts were precipitated by centrifugation (1,000 x g for 2 min)

and resuspended in 30 μl of Renilla Luciferase Assay Lysis Buffer. Then 6 μl of each cell lysate were added to 30 μl of

Renilla Luciferase Assay Reagent and luminescence was immediately measured with a Lumat LB 9507 Tube

Luminometer (Berthold Technologies, NY, USA). For luminescence quantification, five biological replicates were

analyzed for each protein-protein interaction.

Total RNA extraction, qPCR and RNA gel blots. RNA extraction, removal of genomic DNA, cDNA synthesis and

qRT-PCR analyses were performed as described previously47with a minor modification (omission of aurintricarboxylic

acid) to make the protocol compatible with the subsequent PCR procedures. GAPDH and 40S rRNA were used as

reference genes. Three replicates for each experiment were used and the average expression value was calculated. For a

list of the primers used and designed using QuantPrime (http://quantprime.mpimp-golm.mpg.de/)48 see Supplementary

Table 4. RNA gel blots were performed as previously described42 using probes produced using the primers reported in

Supplementary Table 4.




4

Yeast two-hybrid assay. The ProQuestTM Two-hybrid System and the Easy Comp (Life Technologies) were used. The

CDS of all the genes of interest were amplified and cloned as described above. In addition, the CA and DN mutants of

ROP2 were produced by substituting the amino acids corresponding to CA (G15V substitution) and DN (T20N

substitution) mutants as previously reported20. The mutant genes were then subcloned into pENTR/D-TOPO, as

described above. For RbohD (1-355) the sequence corresponding to the N-terminal region, which encodes the cytosolic

domain containing EF-hand motifs, was amplified using the primers listed in Supplementary Table 2 and then

subcloned into pENTR/D-TOPO, as described above. Each entry vector was then recombined with the activation

domain (AD) vector pDEST22 and/or the binding-domain (BD) vector pDEST32, as dictated by the needs of the

experiment. S. cerevisiae strain Mav203 was transformed with 800 ng each of the different combinations of bait, prey

and control (non-recombined) vectors. Colonies containing both AD and BD vectors were selected by plating at 30 °C

for 3 days on a minimal selective dropout medium lacking Leu and Trp (SC-LW medium). These clones were

subsequently replicated on selective dropout medium (SC-LWUH-3AT medium) lacking Leu, Trp, Uracyl, and His and

supplemented with 50mM 3-aminotriazole (3AT). The interaction was further verified by β-galactosidase staining

(LacZ) following the manufacturer’s instructions. The ULTImate Y2H™ yeast two-hybrid screening reported in Table

1 was performed by Hybrigenics (www.hybrigenics.com). In this case the coding sequence of HRU1 (At3G03270) was

cloned into pB27 as a C-terminal fusion to LexA (N-LexA-prolyl 4-hydroxylase-C) and transformed into yeast. The

Arabidopsis (Col-0) 1-week-old seedling cDNA library RP1 was used. After mating, 101 million clones were screened.

Prey fragments of the positive clones were amplified by PCR and sequenced at 5′ and 3′ junctions. The resulting

sequences were used to identify the corresponding interacting proteins in the GenBank (National Center for

Biotechnology Information) database using a fully automated procedure.

ROP pulldown assay.

The assay was performed as previously described11, using a commercially available antibody against ROP proteins,

including ROP2. The IPTG-inducible form of RIC1-Maltose Binding Protein (MBP) fusion protein was expressed in

Rosetta 2 E. coli cells and extracted using sonication. The pMAL-c2 plasmid carrying the coding sequence for the

fusion protein was kindly provided by Prof. Zhenbiao Yang (University of California, Riverside). Four-week old plants

vertically grown in agar plates were used. Total protein extract from leaves (1g) and roots (1g) were ground in liquid

nitrogen using the extraction buffer as previously reported59, (weight/volume ratio 1:2 for leaves and 1:1 for roots). A

fraction of total proteins was loaded for immunoblotting using an antibody (Rabbit polyclonal to AtRAC3 from

abcam®) which is able to recognize ROP2 as well. As an internal control, extracts from plants transformed with

35S:ROP2 were used to detect the correct band size (not shown). Thirty μl of MBP-RIC1 beads were added to the

protein extract, and volumes were adjusted using the extraction buffer lacking Triton X-10011. The samples were gently

shaken for 3h at 4°C and the pellet was washed three times. Proteins were then fractionated by 10% SDS-PAGE,

transferred to PVDF membrane using Trans-Blot® Turbo™ Transfer System (Biorad ®) and incubated with the anti-

RAC3 antibody (0.5μg/ml), followed by horseradish peroxidase-conjugated anti-rabbit IgG (1:20000). The

chemiluminescent signal was detected using ECL reagent (LiteAblot® TURBO, EuroClone®) and Biospectrum®

Imaging System (UVP®) to detect frames at different exposure times. The best signal was detected after 5 minutes of

exposure for both leaves and roots.

5

Agroinfiltration of Nicotiana benthamiana and detection of ROS. For the agroinfiltration experiments the cds

sequences of HRU1, truncated version of HRU1, wild-version of ROP2, and constitutively activated-version of ROP2,

subcloned into pENTR/D-TOPO as described above, were recombined with the destination vector pK7WG252 to obtain

the corresponding expression vectors carrying the constructs 35S:HRU1, 35S:HRU1tr, 35S:ROP2 and 35S:ROP2-CA.

Agroinfiltration of N. benthamiana leaves was performed as previously described26. ROS were visualized in root hairs

and leaves by 3,3’-diaminobenzydine (DAB) staining50. A quantitative measurement of hydrogen peroxide production

was performed on the external medium of 8-day old liquid-grown seedlings grown under dim light, using the Amplex

Red hydrogen peroxide/peroxidase assay kit (Life Technologies) following the manufacturer’s instructions.

Confocal imaging

In BiFC experiments, YFP fluorescence was analyzed with a Nikon Eclipse Ti-5 ViCo video confocal microscope

(http://www.nikon.com/) using YFP filters. In the other experiments, cell fluorescence was measured using an Olympus

FV100 inverted confocal microscope interfaced with an Argon laser for excitation at 488 nm (for GFP and chloroplasts

imaging) and 514 nm (for YFP imaging). Glass bottom Petri dishes containing transfected cells were mounted on the

microscope and viewed with a 60x1.25 NA water immersion objective. Live cell imaging has been always performed at

room temperature. The following fluorescence collection ranges were adopted: 500-550 nm (GFP), 525-580 nm (YFP),

600-700 nm (chloroplasts).

Fluorescence Recovery After Photobleaching (FRAP) experiments

Fluorescence imaging per se cannot yield quantitative information about the exact protein sub-cellular localization. For

instance, instance, proteins diffusing within the cytoplasm cannot be distinguished from proteins diffusing on the

membrane in the particular case in which they are both enclosed within the sub-micron-thick contour of the cell

(comparable in size with the diffraction-limited observation spot). Each FRAP experiment started with a 2-time line-

averaged image of the entire protoplast (pre-bleach image) followed by a raster-scan bleach of a micron-sized region of

the cell cytoplasm/membrane (typically, a rectangular region with dimensions 10x1μm) with laser pulse at full power

for the minimum time required to photobleach most of nuclear fluorescence. Recovery was then measured by starting a

time lapse acquisition within few milliseconds from the end of bleaching (sampling rate has been tailored to the speed

of fluorescence recovery of the tested protein). Each image of the recovery was 2-time line-averaged. Image size was

256x256 pixels and scan speed was usually set to 8μs/pixel. The pinhole size was set to the optimal value of 1.0 Airy.

Before fitting, the experimental values of fluorescence in the bleached area were normalized by the fluorescence of the

entire cell at the same time (each subtracted of the background noise), in order to minimize the effect of cell motility

and defocusing on the recovery curves and to correct for bleaching caused by imaging. Moreover, data were normalized

by pre-bleach fluorescence values in order to verify the presence of an immobile fraction of fluorescent molecules

within the bleached compartment. Retrieved FRAP curves were fitted to either mono- or double-exponential equations

of the form:

( ) ( )τ/texpAFtF −⋅+= ∞ [1]

( ) ( ) ( )2211 τ/texpAτ/texpAFtF −⋅+−⋅+= ∞ [2]




6

where F(t) is the fluorescence value at time t. The three fitting parameters in each equation refer to the asymptotic

fluorescence (F∞), the dynamic range of recovery (A, or A1 and A2), and the time constant of fluorescence exponential

recovery (τ, τ1, and τ2). In the anoxic treatment experiments the assumption is made that GFP-HRU1 molecules are split

into two populations only (localized to the cytoplasm and membrane, respectively). Consequently, data are fitted to a

double-exponential equation in the form of Eq. 2, where τ1, and τ2 are set to the two characteristic times of GFP-HRU1

diffusion (6.1 ± 0.7 and 60.2 ± 3.7 seconds, respectively) and F∞ is set to 1 (according to the observed absence of

detectable IF for HRU1). A1 and A2 are extracted from fitting, and used to calculate the fractional contribution of each

population, namely: A1/( A1 + A2) for the fast one, and A2/(A1 + A2) for the slow one. On this basis, physiological and

anoxic conditions are quantitatively compared.

Statistical analysis for FRAP experiments

The Coefficient of Determination60 (R2) for the data reported in Figure 4b-e was calculated to verify how well data fit

the used statistical model. Mono-exponential fit is appropriate for GFP (R2=0.99). By contrast, a mono-exponential

interpolation must be rejected for HRU1-GFP (R2<0.9), that is satisfactorily fitted by a double-exponential function

(R2=0.98). For the HRU1-HRU1-YFP in BiFC experiments the mono-exponential fit has R2=0.98. For ROP2-GFP the

mono-exponential fit has R2=0.98. For RbohD-GFP the double-exponential fit has R2=0.97. Concerning the HRU1-GFP

subcellular localization after 6h of anoxic treatment (Figure 4g), the double-exponential fit reveals a relative increase in

the slow-population abundance, from 32% to 52% after 6h of anoxia. The statistical significance of the difference

between the two curves was further checked by a one-tailed Mann-Whitney U test61 conducted between the two

populations of fluorescence intensity values (N=24 values for HRU1-GFP under physiological conditions and N=12

values for HRU1-GFP after 6h of anoxia) measured at t=40 s from bleaching (a time point at which HRU1-GFP

recovery in physiological conditions is almost 90% completed). The test yielded a p-value of 0.043: the null hypothesis

(i.e. the two populations of values have equal distribution) can be therefore rejected.

7

Supplementary References

51. Alonso, J.M. et al. Genome-Wide Insertional Mutagenesis of Arabidopsis thaliana. Science 301, 653-657

(2003).

52. Karimi, M., Inzé, D. &Depicker, A. GATEWAY vectors for Agrobacterium-mediated plant transformation.

Trends Plant Sci. 7, 193-195 (2002).

53. Zhang, X., Henriques, R., Lin, S.S., Niu, Q.W. & Chua, N.H. Agrobacterium-mediated transformation of

Arabidopsis thaliana using the floral dip method. Nat. Protoc. 1, 641-646 (2006).

54. Zhong, S., Lin, Z., Fray, R.G., Grierson, D. Improved plant transformation vectors for fluorescent protein

tagging. Trans. Res. 17, 985-989 (2008).

55. Walter, M. et al. Visualization of protein interactions in living plant cells using bimolecular fluorescence

complementation. Plant J. 40, 428-438 (2004).

56. Weltmeier, F. Combinatorial control of Arabidopsis proline dehydrogenase transcription by specific

heterodimerisation of bZIP transcription factors. EMBO J. 25, 3133-3143 (2006).

57. von Arnim, A.G., Deng, X.W.& Stacey M.G. Cloning vectors for the expression of green fluorescent protein

fusion proteins in transgenic plants. Gene 221, 35-43 (1998).

58. Fujikawa, Y.& Kato, N. Split luciferase complementation assay to study protein-protein interactions in

Arabidopsis protoplasts. Plant J. 52, 185-195 (2007).

59. Xu, T. et al. Cell surface- and Rho GTPase-based auxin signaling controls cellular interdigitation in

Arabidopsis Cell 143, 99-110 (2010).

60. Steel, R. G. D.& Torrie, J. H. Principles and procedures of statistics with special reference to the biological

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Supplementary Figures

Supplementary Fig. 1. Expression of 1MJH-like Universal Stress Proteins under low oxygen conditions and in

genotypes altered in oxygen sensing. A large dataset of microarray analyses performed under various conditions of

low-oxygen (Stress) were selected and queried using Genevestigator. The lower part of the Figure (Genetic

Background) shows the pattern of expression in the genotypes affected in oxygen sensing: 35S:amiRAP2.2-12 (silenced

RAP2.2 and RAP2.12 lines), 35S:HA:RAP2.12 (stable version of RAP2.12), ate1-2 ate 2-1 and prt6 mutants. See Gibbs

et al., 2011, for details on these lines.

9

Supplementary Fig. 2. Induction of HRU1 expression under anoxia in the wild-type (Col-0), hru1-1, and hru1-2.

Expression levels are shown as relative units, with the wild-type value at time 0 set to a value of one. Each value is the

mean (±SD) of three measurements.




10

Supplementary Fig. 3. Phenotypes of wild-type (Col-0), hru1-1, hru1-2, 35S:HRU1 and hru1-1 x 35S:HRU1

plants. a, Leaf phenotype. b, Root hair phenotype. c, Root phenotype. d, Inflorescence phenotype. e, Seedling

phenotype.

11

hru1-1 mutant

Promoter sequence: lower case CDS: upper case ATG=ATG Exons: GREEN DELETED SEQUENCES: AAGGTAAA

tactataattttttgtaaacaactcgtaagtcgtatataaaccatttaaacttcgcttgaaaaatcagagtaagctagtccaatttaaacgatatttaagcactagaatcataacggaaagatctttttatattgttagtgtaactaaaagatcttcttctagcgaaaattattatttcctttttatttcttgtatatttacaaaactctgaaacccgttagaggcttcaccaagattcaagcatcaaagccactatatatatatcactcacacttgttaattaattatcagtatttgtttcttcatcatgatgaaaccattttctctagtactcgttcttgtcctctcatcatctttagcaagtgcaaccgcatcctccttattctcgcctccacttccattttcgctctcttccatcttcggtgggattatcggtaacagtgataatagcgagttagctaggatgtgttttccagacctaggagacggagaagcctgcgtggcggaaatcttcggttcattctttagtcctcaaattacgataggaccagaatgttgcaaggccattgttgagatcgacgaagactgtgctcaagccatttttaaaccactcagcaattctttctttagtagttctgtcaagcagtattgcacttacatcaatagttgattgaattttctttaggtttactttagtgcttgttttgtttaatgtcttttatttgtttcttgttttctatgttttatcaacactcaacagtaacttcattttaataagtttaaacaaaattttgaattggaataatatttcaccgaaggtgttggtacgtgttccatcaatcaatgaataaaaaaaaataagccaataaaggaagaaaagttgaagaatcaaattcgcctttgctgaaagttgacaaagaatattacttttcttggagagcaagtgagataaagagaattcactaatactctacgtgtgcaaatacatatcttgaatcttctatggctttcatcagtacacaatttaagatttcgactaaaagaaaaccaaaatgtattcgctcacatcactttaaaatcatgttttcaatatcaagtatattaaataattcttttcaaactatatgaggttactgaaatgttatgcacacaaaagaaataaaaagacaaaactattcaaaagaaatgactagtcattaatactctcagtaacaaagaattttaagaaaagaagcgagagagagagacctagtagtttcaagaatctccccatagaagaaaaagacaatcattaaatatctcgaattctgtgtgcgccaataatattctccaaagtctaaaccatcaaaccggttattatatacatataaattagactaccgtaatcaatttctacttagagatcatcgagagggcaagagagaaaa ATGGGAAAGGCACGTACGGTGGGAGTGGGAATGGACTATTCTCCGACGAGCAAATTAGCTCTCCGGTGGGCGGCGGAGAATCTCCTTGAAGACGGCGACACCGTCATCTTGATTCACGTCCAACCACAAAACGCCGATCATACCCGCAAAATCCTCTTCGAGGAAACCGGTTCACGTATTTAACTTACTACATCTCTTTAATATATATTAGTAGAGTTAACTCCATATTATGATCAAAATTTGTATTTTGATCATTTAAAAGTTACAAACTAATTATTGCAGCGCTAATTCCTTTGGAGGAATTTAGAGAGGTTAATTTGTCTAAACAGTATGGACTTGCTTACGATCCTGAGGTTCTTGATGTTCTTGATACTCTCTCTAGGGCTAAAAAGGTAAATGTTCACTTTCGTAATTCGTAATCTTACACAAGTTAACAACTATTGCTTTAATATCATTTTGATTTTCTTTCAAATAATGATGTTTAATTCATTGTAATATAAACTTTAAACAAAATTATAGGTGAAGGTTGTAGCAAAGGTGTATTGGGGAGATCCAAGGGAGAAACTTTGTGATGCTGTCGAAAATCTAAAACTCGATTCCATTGTTCTTGGCAGTCGAGGTTTGGGTTCLB-T-DNA-LB (TCTCAAAAGGTAAATTCTTATGCTT) ACTTACTATATATAGTCTAGTTTCGTTGGTTAAAAAATTTCGAATTTCAGTAAATTTAGGCAATATAGCTTCAAATAATTGAAACTGATTTGGCTTTTCTCAAATTAATTTTAGTATCACTTATTTCGTTATTCTGTGGTTAATCAAAAGGAGAATTGGAGTGATAATTAATAATTACGTTATCTGTGGGGTTTTTTGTTGTTGTTGTAGGATCTTGCTGGGTAGTGTGAGCAATCATGTGGTGACAAATGCAACATGTCCTGTCACAGTTGTTAAGGCTAATTAAAGTGTCTCTTGTTCTTTAATTGGACCCAGTTAATAACATTAGTAGTTACGTGTGTCACTCATGCATTGTTTTGACATTTATACGTTTGTTGCCTTCTACAAATTTGTGTGAAACTGATTGGTACTGTTTCATTAATAAATTTCAGTTAAATGATATTTACTCTCTTTTTGTTTGTGATTTGTGAACGTTACGTAAAAATCACAAAAAAAAATAATTAGTTTTTCTAATTCATTCATGAAAAATCTTCGTGGACTAAACAAAAAAGATGTGAAAAAACAATCCCCATGTTTGATCCAGATTAATTCGATCCGTTAATCAAAAGATGGTCTTAATGAGCATAATTCGGGGATAAAGTCGAATATAGAAATAGTTGTTGAAAAGAGAAATGTTTGGATTGAATCTTGTCAGCTTGATGTGTGGTTGATCACATGACCAATCTATGTATGGAATATGGATGTCGTGTGTGCTGATTCATTCAGATATGACAAGTGGTGGATGGGATAATTCACGATATGGGCCAGATTATGAGTTTGTTTTAACTTGAATGGCAGGCCGAATCATAATGGATTCATGATCATTATGTTTGATCATAGCCTAGTTGGACTTGTATCCCTAGATCATGATCAAAGTGGGCTTATAAACATGGCCGAGATGGGTAGTTCTAAATCAATTTGTTGGATCAGAAGAAATCAAGAAAGAGACAGTCGTTGGAGCTTTTCTGATCTGCTTTTGGATGATCACCCATTAGATTGAAAATTAAATTTTTCATTCTCCGCTCGATGGCTGATTTGTAAACTTGTTCCTTCATGATAGAAATTTATGGGCT




10

Supplementary Fig. 3. Phenotypes of wild-type (Col-0), hru1-1, hru1-2, 35S:HRU1 and hru1-1 x 35S:HRU1

plants. a, Leaf phenotype. b, Root hair phenotype. c, Root phenotype. d, Inflorescence phenotype. e, Seedling

phenotype.

11

hru1-1 mutant


tactataattttttgtaaacaactcgtaagtcgtatataaaccatttaaacttcgcttgaaaaatcagagtaagctagtccaatttaaacgatatttaagcactagaatcataacggaaagatctttttatattgttagtgtaactaaaagatcttcttctagcgaaaattattatttcctttttatttcttgtatatttacaaaactctgaaacccgttagaggcttcaccaagattcaagcatcaaagccactatatatatatcactcacacttgttaattaattatcagtatttgtttcttcatcatgatgaaaccattttctctagtactcgttcttgtcctctcatcatctttagcaagtgcaaccgcatcctccttattctcgcctccacttccattttcgctctcttccatcttcggtgggattatcggtaacagtgataatagcgagttagctaggatgtgttttccagacctaggagacggagaagcctgcgtggcggaaatcttcggttcattctttagtcctcaaattacgataggaccagaatgttgcaaggccattgttgagatcgacgaagactgtgctcaagccatttttaaaccactcagcaattctttctttagtagttctgtcaagcagtattgcacttacatcaatagttgattgaattttctttaggtttactttagtgcttgttttgtttaatgtcttttatttgtttcttgttttctatgttttatcaacactcaacagtaacttcattttaataagtttaaacaaaattttgaattggaataatatttcaccgaaggtgttggtacgtgttccatcaatcaatgaataaaaaaaaataagccaataaaggaagaaaagttgaagaatcaaattcgcctttgctgaaagttgacaaagaatattacttttcttggagagcaagtgagataaagagaattcactaatactctacgtgtgcaaatacatatcttgaatcttctatggctttcatcagtacacaatttaagatttcgactaaaagaaaaccaaaatgtattcgctcacatcactttaaaatcatgttttcaatatcaagtatattaaataattcttttcaaactatatgaggttactgaaatgttatgcacacaaaagaaataaaaagacaaaactattcaaaagaaatgactagtcattaatactctcagtaacaaagaattttaagaaaagaagcgagagagagagacctagtagtttcaagaatctccccatagaagaaaaagacaatcattaaatatctcgaattctgtgtgcgccaataatattctccaaagtctaaaccatcaaaccggttattatatacatataaattagactaccgtaatcaatttctacttagagatcatcgagagggcaagagagaaaa ATGGGAAAGGCACGTACGGTGGGAGTGGGAATGGACTATTCTCCGACGAGCAAATTAGCTCTCCGGTGGGCGGCGGAGAATCTCCTTGAAGACGGCGACACCGTCATCTTGATTCACGTCCAACCACAAAACGCCGATCATACCCGCAAAATCCTCTTCGAGGAAACCGGTTCACGTATTTAACTTACTACATCTCTTTAATATATATTAGTAGAGTTAACTCCATATTATGATCAAAATTTGTATTTTGATCATTTAAAAGTTACAAACTAATTATTGCAGCGCTAATTCCTTTGGAGGAATTTAGAGAGGTTAATTTGTCTAAACAGTATGGACTTGCTTACGATCCTGAGGTTCTTGATGTTCTTGATACTCTCTCTAGGGCTAAAAAGGTAAATGTTCACTTTCGTAATTCGTAATCTTACACAAGTTAACAACTATTGCTTTAATATCATTTTGATTTTCTTTCAAATAATGATGTTTAATTCATTGTAATATAAACTTTAAACAAAATTATAGGTGAAGGTTGTAGCAAAGGTGTATTGGGGAGATCCAAGGGAGAAACTTTGTGATGCTGTCGAAAATCTAAAACTCGATTCCATTGTTCTTGGCAGTCGAGGTTTGGGTTCLB-T-DNA-LB (TCTCAAAAGGTAAATTCTTATGCTT) ACTTACTATATATAGTCTAGTTTCGTTGGTTAAAAAATTTCGAATTTCAGTAAATTTAGGCAATATAGCTTCAAATAATTGAAACTGATTTGGCTTTTCTCAAATTAATTTTAGTATCACTTATTTCGTTATTCTGTGGTTAATCAAAAGGAGAATTGGAGTGATAATTAATAATTACGTTATCTGTGGGGTTTTTTGTTGTTGTTGTAGGATCTTGCTGGGTAGTGTGAGCAATCATGTGGTGACAAATGCAACATGTCCTGTCACAGTTGTTAAGGCTAATTAAAGTGTCTCTTGTTCTTTAATTGGACCCAGTTAATAACATTAGTAGTTACGTGTGTCACTCATGCATTGTTTTGACATTTATACGTTTGTTGCCTTCTACAAATTTGTGTGAAACTGATTGGTACTGTTTCATTAATAAATTTCAGTTAAATGATATTTACTCTCTTTTTGTTTGTGATTTGTGAACGTTACGTAAAAATCACAAAAAAAAATAATTAGTTTTTCTAATTCATTCATGAAAAATCTTCGTGGACTAAACAAAAAAGATGTGAAAAAACAATCCCCATGTTTGATCCAGATTAATTCGATCCGTTAATCAAAAGATGGTCTTAATGAGCATAATTCGGGGATAAAGTCGAATATAGAAATAGTTGTTGAAAAGAGAAATGTTTGGATTGAATCTTGTCAGCTTGATGTGTGGTTGATCACATGACCAATCTATGTATGGAATATGGATGTCGTGTGTGCTGATTCATTCAGATATGACAAGTGGTGGATGGGATAATTCACGATATGGGCCAGATTATGAGTTTGTTTTAACTTGAATGGCAGGCCGAATCATAATGGATTCATGATCATTATGTTTGATCATAGCCTAGTTGGACTTGTATCCCTAGATCATGATCAAAGTGGGCTTATAAACATGGCCGAGATGGGTAGTTCTAAATCAATTTGTTGGATCAGAAGAAATCAAGAAAGAGACAGTCGTTGGAGCTTTTCTGATCTGCTTTTGGATGATCACCCATTAGATTGAAAATTAAATTTTTCATTCTCCGCTCGATGGCTGATTTGTAAACTTGTTCCTTCATGATAGAAATTTATGGGCT




12

hru1-2 mutant


tactataattttttgtaaacaactcgtaagtcgtatataaaccatttaaacttcgcttgaaaaatcagagtaagctagtccaatttaaacgatatttaagcactagaatcataacggaaagatctttttatattgttagtgtaactaaaagatcttcttctagcgaaaattattatttcctttttatttcttgtatatttacaaaactctgaaacccgttagaggcttcaccaagattcaagcatcaaagccactatatatatatcactcacacttgttaattaattatcagtatttgtttcttcatcatgatgaaaccattttctctagtactcgttcttgtcctctcatcatctttagcaagtgcaaccgcatcctccttattctcgcctccacttccattttcgctctcttccatcttcggtgggattatcggtaacagtgataatagcgagttagctaggatgtgttttccagacctaggagacggagaagcctgcgtggcggaaatcttcggttcattctttagtcctcaaattacgataggaccagaatgttgcaaggccattgttgagatcgacgaagactgtgctcaagccatttttaaaccactcagcaattctttctttagtagttctgtcaagcagtattgcacttacatcaatagttgattgaattttctttaggtttactttagtgcttgttttgtttaatgtcttttatttgtttcttgttttctatgttttatcaacactcaacagtaacttcattttaataagtttaaacaaaattttgaattggaataatatttcaccgaaggtgttggtacgtgttccatcaatcaatgaataaaaaaaaataagccaataaaggaagaaaagttgaagaatcaaattcgcctttgctgaaagttgacaaagaatattacttttcttggagagcaagtgagataaagagaattcactaatactctacgtgtgcaaatacatatcttgaatcttctatggctttcatcagtacacaatttaagatttcgactaaaagaaaaccaaaatgtattcgctcacatcactttaaaatcatgttttcaatatcaagtatattaaataattcttttcaaactatatgaggttactgaaatgttatgcacacaaaagaaataaaaagacaaaactattcaaaagaaatgactagtcattaatactctcagtaacaaagaattttaagaaaagaagcgagagagagagacctagtagtttcaagaatctccccatagaagaaaaagacaatcattaaatatc LB-T-DNA-LB (tcgaattctgtgtgcgccaataatattctcc) aaagtctaaaccatcaaaccggttattatatacatataaattagactaccgtaatcaatttctacttagagatcatcgagagggcaagagagaaaa ATGGGAAAGGCACGTACGGTGGGAGTGGGAATGGACTATTCTCCGACGAGCAAATTAGCTCTCCGGTGGGCGGCGGAGAATCTCCTTGAAGACGGCGACACCGTCATCTTGATTCACGTCCAACCACAAAACGCCGATCATACCCGCAAAATCCTCTTCGAGGAAACCGGTTCACGTATTTAACTTACTACATCTCTTTAATATATATTAGTAGAGTTAACTCCATATTATGATCAAAATTTGTATTTTGATCATTTAAAAGTTACAAACTAATTATTGCAGCGCTAATTCCTTTGGAGGAATTTAGAGAGGTTAATTTGTCTAAACAGTATGGACTTGCTTACGATCCTGAGGTTCTTGATGTTCTTGATACTCTCTCTAGGGCTAAAAAGGTAAATGTTCACTTTCGTAATTCGTAATCTTACACAAGTTAACAACTATTGCTTTAATATCATTTTGATTTTCTTTCAAATAATGATGTTTAATTCATTGTAATATAAACTTTAAACAAAATTATAGGTGAAGGTTGTAGCAAAGGTGTATTGGGGAGATCCAAGGGAGAAACTTTGTGATGCTGTCGAAAATCTAAAACTCGATTCCATTGTTCTTGGCAGTCGAGGTTTGGGTTCTCTCAAAAGGTAAATTCTTATGCTTACTTACTATATATAGTCTAGTTTCGTTGGTTAAAAAATTTCGAATTTCAGTAAATTTAGGCAATATAGCTTCAAATAATTGAAACTGATTTGGCTTTTCTCAAATTAATTTTAGTATCACTTATTTCGTTATTCTGTGGTTAATCAAAAGGAGAATTGGAGTGATAATTAATAATTACGTTATCTGTGGGGTTTTTTGTTGTTGTTGTAGGATCTTGCTGGGTAGTGTGAGCAATCATGTGGTGACAAATGCAACATGTCCTGTCACAGTTGTTAAGGCTAATTAAAGTGTCTCTTGTTCTTTAATTGGACCCAGTTAATAACATTAGTAGTTACGTGTGTCACTCATGCATTGTTTTGACATTTATACGTTTGTTGCCTTCTACAAATTTGTGTGAAACTGATTGGTACTGTTTCATTAATAAATTTCAGTTAAATGATATTTACTCTCTTTTTGTTTGTGATTTGTGAACGTTACGTAAAAATCACAAAAAAAAATAATTAGTTTTTCTAATTCATTCATGAAAAATCTTCGTGGACTAAACAAAAAAGATGTGAAAAAACAATCCCCATGTTTGATCCAGATTAATTCGATCCGTTAATCAAAAGATGGTCTTAATGAGCATAATTCGGGGATAAAGTCGAATATAGAAATAGTTGTTGAAAAGAGAAATGTTTGGATTGAATCTTGTCAGCTTGATGTGTGGTTGATCACATGACCAATCTATGTATGGAATATGGATGTCGTGTGTGCTGATTCATTCAGATATGACAAGTGGTGGATGGGATAATTCACGATATGGGCCAGATTATGAGTTTGTTTTAACTTGAATGGCAGGCCGAATCATAATGGATTCATGATCATTATGTTTGATCATAGCCTAGTTGGACTTGTATCCCTAGATCATGATCAAAGTGGGCTTATAAACATGGCCGAGATGGGTAGTTCTAAATCAATTTGTTGGATCAGAAGAAATCAAGAAAGAGACAGTCGTTGGAGCTTTTCTGATCTGCTTTTGGATGATCACCCATTAGATTGAAAATTAAATTTTTCATTCTCCGCTCGATGGCTGATTTGTAAACTTGTTCCTTCATGATAGAAATTTATGGGCT

Supplementary Fig. 4. Schematic representation of the T-DNA insertion in hru1-1 and hru1-2 mutants and

sequencing data showing the T-DNA insertion position

13

Supplementary Fig. 5. 3,3’-diaminobenzydine (DAB) staining of Col-0 and hru1-1 rosettes grown in soil. The

histogram shows the staining intensity quantitation by ImageJ. Data are mean of 10 measurements, ± SD. Statistical

significance was determined using one-way ANOVA as compared to Col-0, where *p<0.05.




14

Supplementary Fig. 6. Expression of Rboh genes under low oxygen conditions and in genotypes altered in oxygen

sensing. A large dataset of microarray analyses performed under various conditions of low-oxygen (Stress) were

selected and queried using Genevestigator. The lower part of the figure (Genetic Background) shows the pattern of

expression in the genotypes affected in oxygen sensing: 35S:amiRAP2.2-12 (silenced RAP2.2 and RAP2.12 lines),

35S:HA:RAP2.12 (stable version of RAP2.12), ate1-2 ate 2-1 and prt6 mutants. See Gibbs et al., 2011, for details of

these lines.

15

Supplementary Fig. 7. Controls for the Bimolecular Fluorescence complementation experiments reported in Fig.

3. Arabidopsis mesophyll protoplasts were transformed with the amount of plasmid DNA indicated in the figure. The

amount of DNA used for each couple of plasmids is the lowest that gave a signal, as shown in Fig. 3, while not

displaying significant fluorescence in the respective control [with the exception of the interaction RbohD-YFPN +

YFPC-HRU1 (5 µg per each construct) which did not give a signal (Fig. 3b) differently from one of its control (-YFPN +

YFPC-HRU1)].




16

Supplementary Fig. 8. a. ROP2-GTP and total ROP levels in roots and leaf extracts from WT and hru1-1 4-week-old

plants grown on vertical agar plates. The immunoblot shows the levels of total ROP (ROP-GTP and ROP-GDP) in

crude extracts or ROP-GTP after pull-down with RIC1-maltose binding protein. Data are from the three independent

experiments shown. b. Densitometric analysis of the immunoblots shown in panel a.

17

Supplementary Fig. 9. 3,3’-diaminobenzydine (DAB) staining of Nicotiana benthamiana leaves transiently

transformed with β-glucuronidase (35S:GUS), HRU1 (35S:HRU1), truncated version of HRU1 (35S:HRU1tr), wild-

version of ROP2 (35S:ROP2), constitutively activated-version of ROP2 (35S:ROP2-CA). Combinations of constructs

are shown in the graph legend. Data are from three independent experiments, as shown in the graph legend (Exp.1, Exp.

2, Exp.3). The DAB-staining intensity of independent replicates (from 10 to 16, as shown in the graph legend) was

measured densitometrically from images of the Nicotiana leaves. The intensity obtained with 35S:GUS was set to 100

and used as a reference to calculate the DAB staining intensity (Y-axis). Significant variations between treatments and

the control were evaluated statistically by one-way ANOVA. Mean values that were significantly different (P<0.05)

from the control (35S:GUS, set to 100) are marked with *.

0

50

100

150

200

250

300

*

** *

Rela

tive

DAB

stai

ning

Inte

nsity




18

Supplementary Fig. 10. Induction of ADH, AHb1, ZAT10, ZAT12 expression under anoxia in the wild-type (Col-

0), hru1-1, 35S:HRU1, hru1-2, and hru1-1x35S:HRU1. Seedlings were grown on vertical plates as described in Fig.

5c. Treatment under anoxia was 8h-long. Expression levels are shown as relative units, with the wild-type value at time

0 (aerobic) set to a value of one. Each value is the mean (±SD) of three measurements. Mean values that were

significantly different (One-way ANOVA, p<0.05) from the WT are marked with *.

19

Supplementary Fig. 11. Expression of HRU1 in the wild-type (Col-0), 35S:HRU1-A, and 35S:HRU1-B. Expression

levels are shown as relative units, with the wild-type value set to a value of one. Each value is the mean (±SD) of three

biological replicates.




20

Supplementary Table 1. Genes interacting with HRU1 (bait) in a yeast two-hybrid screen against an Arabidopsis

seedling (one-week-old) prey library. Predicted Biological Score (PBS) represents the probability of an interaction

being nonspecific. A, Very high confidence in the interaction; C, Good confidence in the interaction; D, Moderate

confidence in the interaction.

Gene Name/Family AGI code PBS Score No. of Interactions

Universal Stress Protein/Hypoxia

responsive Universal Stress

Protein (HRU1)

At3G03270 A 15

Universal Stress Protein AT3G17020 A 218


Protein contains putative RNA

binding domain (RAP)

AT2G31890 C 2

Clathrin, heavy chain AT3G08530 D 5

Insulinase AT5G42390 D 1

21

Supplementary Table 2 Primers used for gene cloning

Gene Forward primer Reverse primer Reverse primer

without stop-codon

HRU1 cds

(At3g03270.2)

CACCATGGGAAAGGCA

CGTACGGTGGGA

TTAATTAGCCTTAACAA

CTGTGACAGGACA

HRU1 genomic

sequence

CACCATGGGAAAGGCA

CGTACGGTG

TCAATCTAATGGGTGA

TCATCCAAA

HRU1tr CACCATGGGAAAGGCA

CGTACGGTG

TCATCGACTGCCAAGA

ACAATGGA

ROP2

(At1g20090)

CACCTTTTTGTTTGTTT

CCGATCTTGC

TCTGCTTCTTTCTTTAG

TTGGTTTTACC

GGGCAAGAACGC

GCAACGGTTC

RbohD

(At5g47910)

CACCATGAAAATGAGAC

GAGGC

CTAGAAGTTCTCTTTGT

GGAAGTCAAAC

GGGGAAGTTCTC

TTTGTGGAAGTC

A

RbohD

(N-terminal

region)

CACCATACACAAAAATC

AAACACCT

TTATCTTCCATTATCAG

TGCCGCATA

TRXh

(At3g51030)

CACCATGGCTTCGGAA

GAAG

TTAAGCCAAGTGTTTG

GCAATG




20

Supplementary Table 1. Genes interacting with HRU1 (bait) in a yeast two-hybrid screen against an Arabidopsis

seedling (one-week-old) prey library. Predicted Biological Score (PBS) represents the probability of an interaction

being nonspecific. A, Very high confidence in the interaction; C, Good confidence in the interaction; D, Moderate

confidence in the interaction.

Gene Name/Family AGI code PBS Score No. of Interactions

Universal Stress Protein/Hypoxia

responsive Universal Stress

Protein (HRU1)

At3G03270 A 15



Protein contains putative RNA

binding domain (RAP)

AT2G31890 C 2

Clathrin, heavy chain AT3G08530 D 5

Insulinase AT5G42390 D 1

21

Supplementary Table 2 Primers used for gene cloning

Gene Forward primer Reverse primer Reverse primer

without stop-codon

HRU1 cds

(At3g03270.2)

CACCATGGGAAAGGCA

CGTACGGTGGGA

TTAATTAGCCTTAACAA

CTGTGACAGGACA

HRU1 genomic

sequence

CACCATGGGAAAGGCA

CGTACGGTG

TCAATCTAATGGGTGA

TCATCCAAA

HRU1tr CACCATGGGAAAGGCA

CGTACGGTG

TCATCGACTGCCAAGA

ACAATGGA

ROP2

(At1g20090)

CACCTTTTTGTTTGTTT

CCGATCTTGC

TCTGCTTCTTTCTTTAG

TTGGTTTTACC

GGGCAAGAACGC

GCAACGGTTC

RbohD

(At5g47910)

CACCATGAAAATGAGAC

GAGGC

CTAGAAGTTCTCTTTGT

GGAAGTCAAAC

GGGGAAGTTCTC

TTTGTGGAAGTC

A

RbohD

(N-terminal

region)

CACCATACACAAAAATC

AAACACCT

TTATCTTCCATTATCAG

TGCCGCATA

TRXh

(At3g51030)

CACCATGGCTTCGGAA

GAAG

TTAAGCCAAGTGTTTG

GCAATG




22

Supplementary Table 3. Primers used to screen mutant plants.

Genotype Primer name Primer sequence

hru1-1 03270F320 CCTTTGGAGGAATTTAGAGAGGTTA

03270R834 CCTTTTGATTAACCACAGAATAACG

LBb1 (T-DNA left border) AACCAGCGTGGACCGCTTGCTG

hru1-2 03270promFW CACCTACTATAATTTTTTGTAAACAACTC

03270promRV TTTTCTCTCTTGCCCTCTCGATGATCTCT


35S::HRU1 AttB1fw GGGACAAGTTTGTACAAAAAAGCAGGCT

AttB2rv GGGACCACTTTGTACAAGAAAGCTGGGT

23

Supplementary Table 4. List of primers used for qPCR experiments and for the production of the probe used in the

Northern Blot experiment (Fig. 2).

Gene Primer name Primer sequence

At3g03270, exon 4a

(qPCR)

sg3g03270Lf CATGGCCGAGATGGGTAGTT

sg3g03270Lr CAGAAAAGCTCCAACGACTGTCT

At3g03270, exon 4b

(qPCR)

sg3g03270Sf TCGAGGTTTGGGTTCTCTCAA

sg3g03270Sr TGACAGGACATGTTGCATTTGTC

At1g77120, ADH ADH-FW ACGGAGTCCTCTTTATCACTATCCC

ADH-RV GATCGAGTCCTACTGAATCTGG

Ahb1 AHB1-FW TTTGAGGTGGCCAAGTATGCA

AHb1-RV TGATCATAAGCCTGACCCCAA

AT3G47340, DIN6 DIN6-FW AAGGTGCGGACGAGATCTTTGG

DIN6-RV ACTTGTGAAGAGCCTTGATCTTGC

At3g03270, HRU1 HRU1-FW ACCGGTTCACCGCTAATTCC

HRU1-RV TCAGGATCGTAAGCAAGTCCATAC

AT1G27730, ZAT10 ZAT10-FW TCTCCGATTCCTCCTTTGTTCG

ZAT10-RV AGATCGCTTACCCTTTGTCCAG

AT5G59820, ZAT12 ZAT12-FW CATCACAACTACTATCACACCAAACTC

ZAT12-RV ATCCACCGTCGACTTGATCT

At1g13440, GAPDH GAPDH-FW GAATCAACGGTTTCGGAAGA




22

Supplementary Table 3. Primers used to screen mutant plants.

Genotype Primer name Primer sequence

hru1-1 03270F320 CCTTTGGAGGAATTTAGAGAGGTTA

03270R834 CCTTTTGATTAACCACAGAATAACG


hru1-2 03270promFW CACCTACTATAATTTTTTGTAAACAACTC

03270promRV TTTTCTCTCTTGCCCTCTCGATGATCTCT


35S::HRU1 AttB1fw GGGACAAGTTTGTACAAAAAAGCAGGCT

AttB2rv GGGACCACTTTGTACAAGAAAGCTGGGT

23

Supplementary Table 4. List of primers used for qPCR experiments and for the production of the probe used in the

Northern Blot experiment (Fig. 2).

Gene Primer name Primer sequence

At3g03270, exon 4a

(qPCR)

sg3g03270Lf CATGGCCGAGATGGGTAGTT

sg3g03270Lr CAGAAAAGCTCCAACGACTGTCT

At3g03270, exon 4b

(qPCR)

sg3g03270Sf TCGAGGTTTGGGTTCTCTCAA

sg3g03270Sr TGACAGGACATGTTGCATTTGTC

At1g77120, ADH ADH-FW ACGGAGTCCTCTTTATCACTATCCC

ADH-RV GATCGAGTCCTACTGAATCTGG

Ahb1 AHB1-FW TTTGAGGTGGCCAAGTATGCA

AHb1-RV TGATCATAAGCCTGACCCCAA

AT3G47340, DIN6 DIN6-FW AAGGTGCGGACGAGATCTTTGG

DIN6-RV ACTTGTGAAGAGCCTTGATCTTGC

At3g03270, HRU1 HRU1-FW ACCGGTTCACCGCTAATTCC

HRU1-RV TCAGGATCGTAAGCAAGTCCATAC

AT1G27730, ZAT10 ZAT10-FW TCTCCGATTCCTCCTTTGTTCG

ZAT10-RV AGATCGCTTACCCTTTGTCCAG

AT5G59820, ZAT12 ZAT12-FW CATCACAACTACTATCACACCAAACTC

ZAT12-RV ATCCACCGTCGACTTGATCT

At1g13440, GAPDH GAPDH-FW GAATCAACGGTTTCGGAAGA




24

GAPDH-RV CTCGGTGGTGATGAAAGGAT

At2g37270,

40SrRNA

40S-FW TCGACGCTGAGATTCAACAG

40S-RV CGTAACCGAAACGTCATCAA

At3g03270, exon 1

(probe for Northern

blot)

03270IEFW GACTATTCTCCGACGAGCAAATTA

03270IERV GAAGAGGATTTTGCGGGTATGAT

At3g03270, exon 4a

(probe for Northern

blot)

03270UEFW ATTATGTTTGATCATAGCCTAGTTGGA

03270UERV ACTGTCTCTTTCTTGATTTCTTCTGAT


universal stress protein hru1 mediates the universal ... · the universal stress protein hru1...

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