Isolation and characterization of rice (Oryza sativa L.)E3-ubiquitin ligase OsHOS1 gene in the modulationof cold stress response

Tiago Lourenco • Helena Sapeta • Duarte D. Figueiredo •

Mafalda Rodrigues • Andre Cordeiro • Isabel A. Abreu •

Nelson J. M. Saibo • M. Margarida Oliveira

Received: 12 August 2012 / Accepted: 11 June 2013

� Springer Science+Business Media Dordrecht 2013

Abstract Plants can cope with adverse environmental

conditions through the activation of stress response sig-

nalling pathways, in which the proteasome seems to play

an important role. However, the mechanisms underlying

the proteasome-mediated stress response in rice are still not

fully understood. To address this issue, we have identified a

rice E3-ubiquitin ligase, OsHOS1, and characterized its

role in the modulation of the cold stress response. Using a

RNA interference (RNAi) transgenic approach we found

that, under cold conditions, the RNAi::OsHOS1 plants

showed a higher expression level of OsDREB1A. This was

correlated with an increased amount of OsICE1, a master

transcription factor of the cold stress signalling. However,

the up-regulation of OsDREB1A was transient and the

transgenic plants did not show increased cold tolerance.

Nevertheless, we could confirm the interaction of OsHOS1

with OsICE1 by Yeast-Two hybrid and bi-molecular

fluorescence complementation in Arabidopsis protoplasts.

Moreover, we could also determine through an in vitro

degradation assay that the higher amount of OsICE1 in the

transgenic plants was correlated with a lower amount of

OsHOS1. Hence, we could confirm the involvement of the

proteasome in this response mechanism. Taken together

our results confirm the importance of OsHOS1, and thus of

the proteasome, in the modulation of the cold stress sig-

nalling in rice.

Keywords Abiotic stress � Proteasome �Cold stress signalling � Transcription factors

Introduction

Abiotic stresses (i.e. cold, drought and salt stress) are major

constrains to agronomical productivity worldwide with low

temperature severely restricting the geographical distribu-

tion of plants (Guy 1990). Plants have evolved mechanisms

to respond and adapt to low temperature through processes

involving membrane stabilization, synthesis and accumu-

lation of specific solutes and changes in enzyme activity, in

a process known as cold acclimation (Thomashow 1999).

However, the molecular mechanisms underlying the

acclimation process are not fully understood and have been

under focus in the past years, aiming to develop more cold-

tolerant crops. The promoter of several cold-stress

responsive genes contains the DRE/CRT (dehydration-

responsive element/C-repeat binding) (CCGAC) cis-motif

(Yamaguchi-Shinozaki and Shinozaki 1994) which is

essential for expression of stress-inducible genes. Specific

transcription factors (TFs), belonging to the AP2/ERF

superfamily, were found to bind and control the expression

of stress-inducible genes with the CRT/DRE cis-motif.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11103-013-0092-6) contains supplementarymaterial, which is available to authorized users.

T. Lourenco � H. Sapeta � D. D. Figueiredo � M. Rodrigues �A. Cordeiro � I. A. Abreu � N. J. M. Saibo � M. M. Oliveira (&)

Genomics of Plant Stress laboratory, Instituto de Tecnologia

Quımica e Biologica, Universidade Nova de Lisboa, Av. da

Republica, 2780-157 Oeiras, Portugal

e-mail: mmolive@itqb.unl.pt

T. Lourenco � H. Sapeta � D. D. Figueiredo � M. Rodrigues �A. Cordeiro � I. A. Abreu � N. J. M. Saibo � M. M. Oliveira

iBET, Apartado 12, 2781-901 Oeiras, Portugal

123

Plant Mol Biol

DOI 10.1007/s11103-013-0092-6

This group of TFs was named DREB1/CBF (Gilmour et al.

1998; Liu et al. 1998; Stockinger et al. 1997). DREB1/CBF

genes are rapidly and transiently up-regulated upon cold-

stress, to which they were found to respond in an ABA-

independent manner.

HOS1 (high expression of osmotically responsive gene 1)

was first identified in Arabidopsis. hos1 mutant plants, which

showed an increased expression of cold-stress associated

genes (Ishitani et al. 1998) like the DREB1/CBF, but did not

show increased cold-tolerance when compared to wild type

(WT) plants. Nevertheless, the mutant plants reach the

same degree of freezing tolerance after a few days of cold

acclimation. hos1 mutant plants also showed early flow-

ering when compared to WT plants. This was reported as

correlated with lower levels of expression of the Flowering

Locus C (FLC) gene (Lee et al. 2001). HOS1 protein was

described as a RING E3 ubiquitin ligase that mediates the

degradation of the ICE1 (Inducer of CBF expression 1)

protein (Chinnusamy et al. 2003) during cold-stress (Dong

et al. 2006). This was the first time that the ubiquitin/pro-

teasome pathway was described as involved in the control

of the cold-stress response. ICE1 is a MYC-like bHLH

(basic Helix-Loop-Helix) transcription factor and it is

thought to be the major responsible for the activation of

DREB1A/CBF3 gene expression (Chinnusamy et al. 2003).

The higher expression of cold stress responsive genes, like

the DREB1A/CBF3, in the hos1 mutant plants is achieved

through the stabilization of the ICE1 protein.

The ICE1 TF is detected at normal growth temperatures

but the DREB1A/CBF3 expression is only transiently

detected after periods of cold treatment. This suggested

that there might be some post-translational modifications to

ICE1 through phosphorylation or SUMOylation. In fact,

the SUMOylation pathway has been implicated in the post-

translational modification of ICE1 through the action of

SIZ1, an E3 SUMO (small ubiquitin-related modifier)

ligase (Miura et al. 2007). SUMOylation is the reversible

conjugation of SUMO proteins to other proteins and can be

involved in several cellular mechanisms, such as tran-

scriptional regulation, apoptosis, protein stability (com-

peting with the ubiquitination pathway) and response to

stress.

In our study, we have isolated the Arabidopsis HOS1

orthologue from rice, which we named OsHOS1. We

observed that OsHOS1 has the RING motif, typical of sev-

eral E3-ubiquitin ligases and we confirmed its E3-ubiquitin

ligase activity. Using a transgenic approach together with

in vitro degradation assays and protein–protein interaction

studies, we showed that OsHOS1 is involved in the cold

signaling and in the OsICE1 proteasome degradation. The

function of the isolated OsHOS1 gene, and the protein pre-

dicted as part of the cold stress response in rice will be

discussed.

Materials and methods

Identification and cloning of the OsHOS1 gene

In order to identify the rice orthologue of the HOS1 from

Arabidopsis (Lee et al. 2001), we have used the BLASTN

tool (Altschul et al. 1997) to search the rice genome. The

homology search retrieved two BAC clones from the same

genome region (OSJNBa0084L17 and OSJNBb0016H12)

with different gene prediction. We have designed primers

(Online Resource 1) with the Primer 3 software (http://

frodo.wi.mit.edu) for the identification of the putative Os-

HOS1 gene using as template rice cDNA, synthesized as

described below (see ‘‘Gene expression analysis’’ section).

The PCR products were sequenced and the longest ORF

was identified as the OsHOS1 gene.

Preparation of the RNA interference (RNAi) genetic

construct

We used the GATEWAY-based (Invitrogen, USA)

pANDA vector (Miki and Shimamoto 2004) to prepare the

RNA interference construct used in this work. A 371 bp

region (primers Fw: GGG GAC AAG TTT GTA CAA

AAA AGC AGG CTTG GTC AAA ATG GTC ACT CAA

AGA and Rv: GGG GAC CAC TTT GTA CAA GAA

AGC TGG GTT CCT CAA ACA AAT CGC AGT TAC A;

underlined regions are the attB regions) from the identified

OsHOS1 sequence was used to prepare the RNAi genetic

construct. The RNAi::OsHOS1 fragment was cloned into

the pDONR221 and, after sequencing confirmation, trans-

ferred to the pANDA vector (Miki and Shimamoto 2004)

and introduced in the appropriate Agrobacterium tum-

efaciens strain (LBA4404).

Production and analysis of transgenic RNAi::OsHOS1

rice plants

For the production of transgenic rice plants, we used a

protocol based on Hiei et al. (1994) with modifications

(Rueb et al. 1994) using rice (Oryza sativa L. cv. Nip-

ponbare) mature seeds. Briefly, embryogenic callus tissues

were selected and co-cultivated with Agrobacterium

LBA4404-RNAi::OsHOS1 strain. The co-cultivated cal-

luses were transferred to selection medium supplemented

with 50 mg/L of Hygromycin B (Duchefa, The Nether-

lands). Hygromycin-resistant callus were transferred to

regeneration medium, and the resistant plantlets potted in

containers with soil mixture (2:2:1, v/v/v, soil:peat:ver-

miculite) and placed in the glasshouse.

The putative transgenic plants were analyzed by PCR

for the presence of the T-DNA insert. Leaf samples from

putative transgenic rice plants were collected, immediately

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123

frozen in liquid nitrogen, and the samples stored at -80 �C

until use. For PCR analysis we used the CTAB protocol to

extract DNA. To assess the integration of the T-DNA

expression cassette in the plant genome, we used the Os-

HOS1 primers (Fw: GGC ACA CTA ACT TAG CAT CTT

GG and Rv: GAG AGG GCT TGA CTT CTT CTG AG)

for the fragment of interest, the hptII gene primers (Fw:

AAT AGC TGC GCC GAT GGT TTC TAC A and Rv:

AAC ATC GCC TCG CTC CAG TCA ATG) for the

selectable marker gene, and the mUbi (Fw: TCT CGA

GAG TTC CGC TCC AC and Rv: ATC TAG AAC GAC

CGC CCA AC) primers for the promoter fragment. Only

the plants with positive PCR amplification for all the

T-DNA components were allowed to grow and self-polli-

nate to retrieve T1 progeny. The PCR-positive transgenic

plants were further analyzed for stable integration of the

transgene by Southern blotting analysis using the promoter

mUbi fragment as probe.

Stress treatment and total RNA extraction

Transgenic and WT plants were germinated in water, in the

dark, at 28 �C. The germinated seedlings were then trans-

ferred to Yoshida’s medium (Yoshida et al. 1976) and

grown for 2 weeks at 28 �C with a 12 h photoperiod and

150–200 lmol/m2/s light intensity (control conditions). For

the cold stress treatment, 2 week-old plants were trans-

ferred to a 10 �C growth chamber with the same photo-

period and light intensity. Whole plant samples were

collected at 0 h (28 �C), 2 h (10 �C), 5 h (10 �C) and 24 h

(10 �C) time points, frozen in liquid nitrogen and stored at

-80 �C until use. A minimum of 4 plants were used per

time point and plant line. Total RNA from whole plants

was extracted using the TRIZOL� (Invitrogen, USA)

reagent following the manufacturer’s protocol.

Gene expression analysis

For cDNA synthesis we treated the total RNA with DNAse

(Qiagen, USA) to eliminate any possible DNA traces. For

the DNAse treatment, the RNA-EZ columns from Qiagen

(USA) were used following the manufacturer’s RNA clean-

up protocol. For cDNA synthesis, we used Invitrogen

(USA) cDNA synthesis kit, following the manufacturer’s

instructions with 1 lg of total RNA and oligo-dT (Invit-

rogen, USA) as primer for first-strand synthesis. For the

target gene expression analysis by semi-quantitative RT-

PCR (sqRT-PCR) we used primers listed as supplementary

material (Online Resource 1). As template for the PCR

reaction we used 2 lL of cDNA (corresponding to 50 ng of

total RNA). PCR was performed using the following con-

ditions: 95 �C for 5 min for denaturing cDNA, 26–28

cycles of 95 �C for 1 min, 53–57 �C for 45 s and 72 �C for

1 min. A final extension period was made at 72 �C for

5 min. In the case of OsDREB1A, 2.5 % of DMSO was

added to the final mix. The rice Ubiquitin-Conjugating

Enzyme E2 (Ubc, LOC_Os02g42314) was used as internal

control. All the RT-PCR experiments were performed

twice (technical replicates) on at least 3 biological

replicates.

For the northern blot analysis (Online Resource 4A),

15 lg of total RNA was electrophoretic separated in a

denaturant RNA gel (19 MOPS buffer, 2 % formaldehyde,

1.2 % agarose) and then transferred to Hybond-N? mem-

brane (Amersham Pharmacia—GE Healthcare, UK) by

capillary blotting. The membrane was probed with the

OsDREB1A (Online Resource 1) fragment using the

Amersham non-radioactive Gene Images plus CDP-Star

chemiluminescent kit (GE-Healthcare, UK) according to

manufacturer’s instructions. As loading control for the

northern blot we used the ethidium bromide staining of

the total RNA/lane. Two biological replicates were used for

the northern blot analysis.

Detection of small interference RNA (siRNA)

in transgenic RNAi plants

We used a protocol described by Goto et al. (2003) to

separate low molecular weight (LMW) RNAs from total

RNA (Goto et al. 2003) with some modifications. 100 lg

of total RNA from transgenic RNAi and WT plants was

used to precipitate LMW RNA’s. The LMW RNAs were

separated in a 15 % polyacrylamide gel (15 % polyacryl-

amide, 7 M urea, 0.5x TBE, 0.1 % APS, 1:2,000 dilution

TEMED) at 200 V for 2.5 h. After electrophoretic sepa-

ration, the LMW RNAs were transferred to Hybond-N?

membrane (Amersham Pharmacia) by capillary blotting.

The RNAs were fixed to the membrane by UV cross-

linking (2 min at 1,200 U plus 2 cycles of 1,200 U) and

baked at 80 �C for 2 h. The membrane was hybridized

using the chemiluminescent kit (GE-Healthcare) using the

RNAi::OsHOS1 fragment as probe following the manu-

facturer’s instructions. As loading control, we hybridized

the membrane with a 5S RNA probe (Online Resource 3).

Rice protoplast isolation and transformation

Rice protoplasts were prepared from etiolated rice seed-

lings (7 to 14-day-old) using a protocol adapted from

others previously described (Bart et al. 2006; Chen et al.

2006). The etiolated shoots and leaves were transferred to a

Digestion solution consisting of 0.4 M mannitol, 10 mM

MES pH 5.7, 1 mM CaCl2, 0.1 % (w/v) BSA, 50 mg/L

ampicillin, 5 mM b-mercaptoethanol, 2.25 % (w/v) Cel-

lulase R10 (Duchefa, The Netherlands) and 0.75 % (w/v)

Macerozyme R10 (Duchefa, The Netherlands). After thin-

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123

slicing the explants (with razor blade), the sections were

vacuum infiltrated for 30 min at room temperature, and

digested for 5 h in the dark with gentle agitation (40 rpm)

at 26 �C. Following enzymatic digestion, the protoplasts

were released with 15 min of mechanical agitation

(80 rpm) at 26 �C and the mixture diluted with one volume

of W5 solution. After filtration through a 50 lm mesh fil-

ter, isolated protoplasts were recovered by centrifugation

for 5 min (3009g) at room-temperature. Protoplasts were

then resuspended in MMg solution (0.4 M Mannitol,

15 mM MgCl2, 4 mM MES pH 5.7), quantified by count-

ing using an haemocytometer, and their concentration

adjusted to 1.0 9 106 cells/mL with MMg solution. For

PEG-mediated transformation, 200 lL of cells were incu-

bated with 10 lg of pANDA-OsHOS1 (1 lg/lL) (or no

plasmid for control) and 220 lL of 40 % (w/v) PEG

solution pH 8.0 (0.4 M Mannitol, 100 mM CaCl2, 40 %

(w/v) PEG MW4000 (Duchefa, The Netherlands)) for

20 min at room-temperature in the dark. PEG solution was

diluted with 3 vol. of W5 solution and the mixture cen-

trifuged as above. The transformed protoplasts were

resuspended in 1.5 mL of W5 solution and transferred to

12-well plates. The transformed cells were incubated for

16–17 h at room-temperature in the dark.

For the cold stress assay, after the 16–17 h incubation,

the plates were transferred to a cold room set to 8–10 �C

for 3 h. Samples were collected at time 0 h (room-tem-

perature) and 3 h (8–10 �C) of cold stress for pANDA-

OsHOS1 and control transformed cells. The collected

samples were centrifuged for 1 min at 7,5009g and the

pellet immediately frozen in liquid nitrogen and stored at

-80 �C until use. Total RNA extraction, cDNA synthesis

and sqRT-PCRs were performed as previously described

above. The gel images were analysed using Image J soft-

ware. At least two independent transformations per tested

condition were used for the analysis.

Total protein extraction and detection of OsICE1

by immunoblot analysis

Total protein extracts were prepared from both

RNAi::OsHOS1 and WT plants, grown at 28 �C, as

described above. Whole 2 week-old plants were collected

or treated for 24 h with 200 lM of a proteasome inhibitor,

MG132 (Sigma, USA). Samples were frozen in liquid

nitrogen and stored at -80 �C until further use. Before

protein extraction, samples were grinded in liquid nitrogen

to a fine powder and allowed to thaw in Protein Extraction

Buffer (PEB; 50 mM Tris–HCl pH 8.0, 150 mM NaCl,

2 mM EDTA pH 8.0, 0.4 % Triton X-100 and 19 com-

plete protease inhibitor (Roche, Switzerland)) (200 lL

per 100 mg of sample powder). The mixture was vortexed

for 2–3 min and kept on ice. Samples were centrifuged

twice at 10,0009g at 4 �C for 15 min to remove cellular

debris. The supernatant was collected and the total protein

was quantified (Bradford method).

Five micrograms of total protein per lane were loaded on

SDS-PAGE and gels were blotted into PVDF membranes.

OsICE1 detection was carried out using a polyclonal

a-OsICE1 (raised in rabbit against two oligopeptides—

EHGQAAPPGQEQHHHC and SPTGQQPRVEVRLREG,

coupled to keyhole limpet hemocyanin (KLH), Eurogentec,

Belgium). Chemiluminescent detection was performed with

Western Lightning Plus-ECL (Perkin-Helmer, USA),

according to manufacturer’s instructions. Coomassie-

stained gels were used as total protein loading control. At

least 2 technical replicates were made from each of the 3

biological replicates. All assays gave similar results.

Expression and purification of recombinant 69HIS-

tagged OsICE1 and OsHOS1

OsICE1 and OsHOS1 coding sequences were cloned in

pDONR221 (Invitrogen, USA) and recombined into

pDEST17 (Invitrogen, USA), in fusion with a 6 9 HIS-tag.

pDEST17/OsICE1 was used to transform BL21 (DE3)

for protein production. Cell were grown to an OD600 of 0.5

and protein expression was induced with 200 lM IPTG

and allowed to occur for 30 min at 37 �C. Cells were

harvested by centrifugation (6,0009g, 45 min, 4 �C). The

bacterial pellet was then resuspended in 20 mM sodium

phosphate pH7.4, 500 mM NaCl, 10 mM imidazole,

500 lM MgCl2, 19 Complete Protease Inhibitor (Roche)

and DNase (1:3 W/V). Cells were lysed in two passages

through a FRENCH Press (1,000 psi). The lysate was

centrifuged for 30 min at 13,0009g and 4 �C. The super-

natant was filtered with a 0.45 lm filter and soluble

6 9 HIS-OsICE1 protein was purified using the His

SpinTrap system (GE Healthcare). Column-bond protein

was eluted with 20 mM TrisHCl pH8, 500 mM NaCl,

250 mM imidazole. Buffer was immediately exchanged to

20 mM TrisHCl pH8, 250 mM NaCl, 1 mM EDTA, 1 mM

DTT and samples were stored at -80 �C. 6 9 HIS-

OsHOS1 was produced similarly as described above.

Ubiquitination assays

For the ubiquitination assay, each reaction (15 lL final

volume) contained 5 lg of HIS-tagged recombinant ubiq-

uitin (U5507, Sigma-Aldrich), 0.05 lg rabbit E1 (E-302,

Boston Biochem, USA), 0.11 lg E2 UbcH5b (E2-622,

Boston Biochem), 300 ng purified HIS-OsHOS1, 2 mM

ATP, 50 mM Tris–HCl (pH 7.4), 5 mM MgCl2, 50 mM KCl

and 1 mM DTT. After incubation at 30 �C for 2 h, the

reaction was stopped by adding 59 SDS-PAGE loading

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buffer. The samples were resolved by electrophoresis on 8 %

SDS-PAGE gels. Ubiquitinated proteins were detected by

western blotting using a-HIS antibody (Abcam, UK).

Degradation assays

Cell-free degradation assays were performed as previously

described (Osterlund et al. 2000). Rice seeds from both WT

and RNAi::OsHOS1 lines were grown for 2 weeks in a

growth chamber in control conditions. The plant material

was collected, immediately frozen in liquid nitrogen and

grounded to a fine powder. Total protein was extracted as

previously described (Osterlund et al. 2000). Five micro-

grams of total protein extracts from WT and RNAi::

OsHOS1 were incubated with an equal amount of

HIS-OsICE1 for 15 and 30 min at 30 �C. To inhibit the

proteasome, we incubated the protein extracts with 200 lM

of MG132 at the start of the assay and collected after

30 min at 30 �C. The degradation reaction was stopped at

each time point by adding 59 SDS-loading buffer. The

reaction products were separated by SDS-PAGE and

transferred into PVDF membrane. Detection of the HIS-

OsICE1 was carried out using a monoclonal a-HIS. Signal

detection was performed as previously described for the

detection of OsICE1. Rice actin was detected as loading

control by western blot (Actin Antibody #sc-1615, Santa

Cruz Biotechnology, USA). Two technical replicates were

made from 3 independent biological replicates. All assays

gave similar results.

Yeast-two hybrid assay

Saccharomyces cerevisiae strain YRG-2 (MATa ura3-52

his3-200 ade2-101 lys2-801 trp1-901 leu2-3 112 gal4-542

gal80-538 LYS2::UASGAL1-TATA GAL1-HIS3

URA3::UASGAL4 17mers(x3)-TATACYC1-lacZ) were

grown in YPD medium supplemented with 20 mg/L of

adenine for the preparation of competent cells through the

LiAc method. Plasmid DNA (pGBK-OsHOS1 and pGAD-

OsICE1) transformation was also performed using the

LiAc method. Yeast transformed cells were plated in

complete minimum (CM) medium lacking leucine and

tryptophan for plasmid transformation control and in CM

lacking leucine, tryptophan and histidine for detection of

interaction. The interaction was evaluated in three indi-

vidual colonies transformed with both plasmids. The

interaction of pAD-WT and pBD-WT from the HybriZAP

2.1 kit (Stratagene, USA) was used as control of positive

interaction. As controls of negative interaction, pGBKT7

and pGADT7 empty vectors were also co-transformed with

pGAD-OsICE1 and pGBK-OsHOS1 respectively.

Bi-molecular fluorescence complementation assays

The coding regions of the OsHOS1 and OsICE1 were

recombined from plasmid pDONR221 into vectors

pYFPN43 and pYFPC43 (Belda-Palazon et al. 2012), to be

in fusion with the N- and C-terminal portions of the yellow

fluorescent protein (YFP), respectively. Cloning was per-

formed using the GATEWAY technology. Arabidopsis

protoplasts were prepared as described (Anthony et al.

2004). For each transformation 3 lg of each plasmid was

used. Transformed protoplasts were incubated for 2 days in

the dark at 22 �C and protein fluorescence observed with a

Leica DM6000B fluorescence microscope. As positive

control, two known interacting proteins were used. As

negative controls in the interaction assays, pYFN-OsHOS1

and pYFC-OsICE1 were co-transformed with pYFC43 and

pYFN43 empty vectors, respectively. More than 50 pro-

toplasts were screened for interaction per transformation.

At least 5 independent transformations were analyzed for

each plasmid combination. The YFP signal was quantified

using the MetaMorph� software version 7.7.0.0 (Molecular

Devices, USA) for 15 protoplasts.

Results

Identification of OsHOS1

By the time this project started, although the rice genome

sequence was already available (IRGSP 2005), the infor-

mation regarding gene organization was still scarce. There-

fore, we used AtHOS1 (NP 181511) amino acid sequence to

BLAST (Altschul et al. 1997) the rice genome searching for

putative OsHOS1 proteins. Two different gene products

were identified with high similarity to AtHOS1, and located

in the same genomic region. Aiming to clarify if there were

two different genes or only one, we designed primers for each

of the predicted rice genes and used them in different com-

binations in RT-PCR (Online Resource 2A). From our study

one single gene with a 2,829 bp open reading frame was

identified and named OsHOS1 (GenBank accession no.

JQ866627, LOC_Os03g52700). OsHOS1 encodes a protein

of 942 amino acids (Online Resource 2B) with a predicted

molecular weight of 106.1 kDa and a predicted isoelectric

point of 6.29. OsHOS1 amino acid sequence is 49 % iden-

tical to AtHOS1 and, similarly to the Arabidopsis pro-

tein, has a modified C3HC4 RING-finger domain in the

N-terminal and a nuclear localization signal (NLS) in

the C-terminal (Fig. 1). Recently, within the same locus

(LOC_Os03g52700), we could identify a similar sequence

which, to our knowledge, was not yet experimentally

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confirmed. The new predicted protein sequence present

in the databases (TIGR, Gramene) is shorter than the

sequence we experimentally describe in Online Resource

2C.

OsHOS1 is an E3-ubiquitin ligase

Aiming to characterize OsHOS1, and since this protein has

the typical RING-motif (Freemont 1993; Joazeiro and

Weissman 2000) present in many E3-ubiquitin ligases (Lee

and Kim 2011; Lyzenga and Stone 2012), we investigated

whether it has an E3-ubiquitin ligase activity as observed

for the Arabidopsis HOS1 (Dong et al. 2006). The OsHOS1

coding sequence was fused in frame with a 6xHIS tag and

expressed in E. coli. The HIS-OsHOS1 protein was purified

from crude extracts through affinity columns and used in

in vitro ubiquitination assays. We could only detect an

ubiquitination signal in the lane where the mixture con-

taining all the necessary components (E1, E2, E3—

OsHOS1, Zn, ATP and Ubq) was run (Fig. 2, lane 2). This

ubiquitination signal was stronger above the predicted MW

of HIS-OsHOS1, which corresponds to the ubiquitinated

forms of HIS-OsHOS1. Taken together, our results show

that OsHOS1 can function in vitro as an E3-ubiquitin

ligase.

RNA interference (RNAi) silencing of OsHOS1

expression

To evaluate the influence of OsHOS1 in rice, we prepared a

genetic construct to silence the expression of OsHOS1

Fig. 1 Sequence alignment of the OsHOS1 and Arabidopsis HOS1 (NP 181511) proteins. The variant C3HC4 RING-finger motif is underlined

with the conserved zinc-binding residues being double underlined. The putative nuclear localization signal (NLS) is indicated with asterisks

Fig. 2 Ubiquitination assay for OsHOS1. HIS-OsHOS1 was assayed

for E3 ubiquitination activity in the presence of E1 activating enzyme,

E2 conjugating enzyme and Ubiquitin. Samples were resolved on 8 %

SDS-PAGE and then blotted on PVDF membrane. a-HIS was used to

detect ubiquitinated forms of HIS-OsHOS1. The plus (?) and the

minus (-) symbols indicate the presence or absence, respectively, of

the specific component in the reaction mixture. As control, HIS-

OsHOS1 alone was used (lane 8)

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using a RNAi vector (pANDA) (Miki and Shimamoto

2004) and produced the rice transgenic line RNAi::

OsHOS1. This line was analyzed for the presence of small

interference RNA (siRNAs) for the OsHOS1 transcript by

northern blot analysis. We were able to detect 2 hybrid-

ization signals below the 25 nt band probably corre-

sponding to the typical 21–24 nt siRNAs mediating gene

silencing (Hamilton et al. 2002) in the RNAi::OsHOS1

line (Online Resource 3). Furthermore, we analyzed

RNAi::OsHOS1 and wild-type plants (WT) for the pres-

ence of OsHOS1 expression under control and stress con-

ditions (10 �C) (Fig. 3a). In WT, OsHOS1 showed a

constitutive expression in all tested conditions, while in the

RNAi::OsHOS1 plants, only a low gene expression level

was detected (both in control and stress conditions).

In order to evaluate cold stress tolerance, we have per-

formed survival and electrolyte leakage studies in rice

plants subjected to cold treatment (10 �C) and cold accli-

mation (temperature decrease from 28 to 6 �C along

14 days), respectively. Despite having performed several

assays, these did not reveal significant differences between

RNAi::OsHOS1 and WT plants (data not shown).

Effect of OsHOS1 silencing on target gene expression

To further investigate OsHOS1 function, we analyzed the

expression profile of selected genes by sqRT-PCR in the

transgenic and WT plants under control and cold (10 �C)

stress conditions (Fig. 3a). The selected genes were puta-

tive rice orthologues previously shown to act together with

AtHOS1 in cold signalling, such as OsICE1 and

OsDREB1A (Dubouzet et al. 2003). In order to assess the

effectiveness of the cold treatment, we included

OsDREB1B in the sqRT-PCR expression analyses, as it

was previously shown to be highly induced by cold

(Dubouzet et al. 2003; Figueiredo et al. 2012). The semi-

quantitative analyses for OsICE1 did not reveal changes in

expression due to cold, neither in the silencing line

(RNAi::OsHOS) nor in the WT plants under the tested

conditions (Fig. 3a). These results are in agreement with

the hypothesis of OsHOS1 being an E3-ubiquitin ligase,

thus not affecting the transcription of this gene but instead

acting at post-translational level controlling the protein

levels. This observation for OsICE1 regulation in rice is

similar to what was found for Arabidopsis (Dong et al.

2006). Regarding OsDREB1A gene expression, it is known

to be rapidly induced under cold stress and the gene is the

closest homologue to AtDREB1A. In our experiments, after

2 h of cold OsDREB1A expression was higher in

RNAi::OsHOS1 plants, than in WT (Fig. 3a). Also, we

could observe that OsDREB1A transcript accumulation was

transient and, after 5 h of cold, the transcript level was

almost undetectable in both WT and silencing lines. The

higher cold stress induction of OsDREB1A in the RNAi as

compared to WT was further confirmed by northern blot

(Online Resource 4A). Together, these results show that

OsHOS1 expression levels affect the rice cold signalling

pathway. We then asked whether the observed

OsDREB1A

up-regulation in the transgenic line could affect the

expression of P5CS, a downstream target gene of

OsDREB1A that contains DRE elements in its promoter

region (Gilmour et al. 2000). P5CS gene encodes a

D-pyrroline 5-carboxylase synthetase which is involved

in proline biosynthesis and osmotic adjustment under

cold acclimation. The expression pattern of this gene can

be used to evaluate the OsDREB1 genetic circuit, and to

distinguish between cold tolerant and sensitive rice lines

(Morsy et al. 2005; de los Reyes et al. 2003). Our results

show that, after cold stress, the P5CS expression is higher

in the OsHOS1 silenced line as compared to the WT

(Fig. 3a). The induction of P5CS correlates with the

Fig. 3 Transcriptional profile of selected genes in WT and OsHOS1

silencing lines under control and cold stress (10 �C) conditions.

A. The gene expression analysis for OsHOS1, OsICE1

(LOC_Os11g32100), OsDREB1A (LOC_Os09g35030), P5CS

(LOC_Os05g38150), OsDREB1B (LOC_Os09g35010) (as cold stress

assay control) was performed by semi quantitative-PCR. The specific

primers can be found in Online Resource 1. Gene expression profile

of Ubiquitin-Conjugating Enzyme E2 (Os02g42314) was used as

internal control for the sqRT-PCR. B. Relative expression changes of

OsDREB1A in rice protoplasts transiently silencing OsHOS1. The

mean expression value of control 0 h was normalized to 1 and the

other mean values represent fold change in expression. The asterisk

(*) represents significantly statistical difference between OsHOS1 3 h

and control 3 h (p \ 0.05) using Student’s t test analysis

Plant Mol Biol

123

expression peak of OsDREB1A after 2 h of cold stress

(Fig. 3a).

Aiming to further confirm the effect of OsHOS1 in the

rice cold response pathway, we used a rice protoplast

system to analyze its function. We transiently silenced the

expression of OsHOS1 with the same vector used to pro-

duce the RNAi line (pANDA-OsHOS1). The transformed

protoplasts were incubated in the dark at 8–10 �C and time

points were collected at 0 h (room-temperature) and 3 h

(8–10 �C). The OsHOS1 gene expression was analyzed to

assess the transformation efficiency. We observed that

OsHOS1 expression level was similarly reduced (*50 %)

in both time points collected (Online Resource 4C). In

addition, and in agreement with the results obtained with

the OsHOS1 silencing line, the protoplasts silencing

OsHOS1 showed a higher induction of the OsDREB1A

expression as compared to control protoplasts after 3 h of

cold stress (Fig. 3b, Online Resource 4B for gel image).

These results, together with the analyses of the OsHOS1

silencing line under cold stress, demonstrate a clear func-

tion of the OsHOS1 in the cold signalling pathway.

OsICE1 accumulates in RNAi::OsHOS1 plants

AtHOS1 was shown to act as an E3-ubiquitin ligase in

Arabidopsis, controlling the levels of AtICE1, a master

transcription factor of the cold signalling pathway

(Chinnusamy et al. 2003; Dong et al. 2006). To know

whether a similar mechanism is maintained or not in rice,

we have analysed OsICE1 protein levels and the effect of

the proteasome inhibitor (MG132) on such levels, in

RNAi::OsHOS1 and WT plants. In immunoblot assays, we

could detect a higher accumulation of OsICE1 in the

RNAi::OsHOS1 as compared to WT plants under control

conditions (Fig. 4). The molecular size of the detected

band was in accordance with the predicted molecular

weight for OsICE1 and in agreement with previous results

in rice (Nakamura et al. 2011). Moreover, we could also

detect a higher accumulation of OsICE1 when the plants

were treated with MG132 for both transgenic and WT

plants (Fig. 4). These results suggest that a lower expres-

sion of OsHOS1 in the transgenic plants leads to a lower

accumulation of the corresponding protein, thus promoting

OsICE1 accumulation. Taken together, this indicates that

OsHOS1 may act as part of the proteasomal complex

regulating the pool of OsICE1 in rice plants. The accu-

mulation of OsICE1 in the transgenic plants may explain

the observed accumulation of OsDREB1A (Fig. 3a), since

in Arabidopsis ICE1 is a positive regulator of DREB1A

(Chinnusamy et al. 2003). In rice, OsICE1 seems to

mediate OsHOS1 effect on cold signalling.

OsHOS1 interacts with OsICE1

In Arabidopsis it was previously shown that HOS1 can

physically interact with ICE1 thus regulating the cold stress

response. Since we observed a higher accumulation of

OsICE1 in the transgenic plants, it was important to verify

if OsHOS1 and OsICE1 could directly interact. To test this

interaction, we used the Yeast-Two hybrid system (Y2H)

and BiFC (Bi-molecular Fluorescence Complementation).

We could demonstrate OsHOS1/OsICE1 interaction by

Y2H, since only the combination of the bait plasmid

pGBK-OsHOS1 with the prey plasmid pGAD-OsICE1 was

able to activate transcription of the auxotrophic marker

gene (HIS3), allowing the cells to grow on selective

medium (Fig. 5).

The interaction was further confirmed by BiFC.

OsHOS1 and OsICE1 sequences were fused into BiFC

plasmids and co-transformed into Arabidopsis protop-

lasts, for transient protein expression. Reconstituted YFP

fluorescence could be observed in the nucleus of pro-

toplasts co-transformed with pYFN-OsHOS1 and pYFC-

OsICE1 (Fig. 6) after 2 days incubation at 22 �C. As

negative control, pYFN-OsHOS1 and pYFC-OsICE1

were co-transformed with pYFC43 and pYFN43 empty

vectors, respectively. No YFP fluorescence was observed

in the negative control in the same conditions as descri-

bed previously.

Taken together, the Yeast-Two hybrid and the BiFC

results suggest a physical interaction between OsHOS1 and

OsICE1.

OsHOS1 is responsible for OsICE1 in vitro degradation

The above results suggest that OsHOS1 acts in a similar

way as to AtHOS1. However, we wanted to further confirm

that OsICE1 would be differentially degraded in an in vitro

cell-free degradation assay (Osterlund et al. 2000) using

crude protein extracts from transgenic and WT plants. To

Fig. 4 OsICE1 protein level in RNAi::OsHOS1 and WT plants. Total

protein from the two plant lines under control conditions or treated

with MG132, was resolved on 10 % SDS-PAGE and then blotted on

PVDF membrane. After blotting, the OsICE1 was detected with the

appropriate antibody (see ‘‘Materials and methods’’). As loading

control, the same amount of total protein used in the immunoblot

assay was resolved on 10 % SDS-PAGE and stained with Coomassie

Brilliant Blue

Plant Mol Biol

123

Fig. 5 Direct Yeast-Two hybrid interaction between OsHOS1 and

OsICE1. The OsHOS1 and OsICE1 sequences were cloned into

pGBKT7 (BD) and pGADT7 (AD) respectively and co-transformed

in the yeast strain YRG2. The interaction was evaluated in selection

CM plates (-HIS-LEU-TRP). The interaction of pADWT and pBDWT

(Stratagene, USA) was used as positive control. As negative controls,

empty pGADT7 and pGBKT7 vectors were co-transformed with

pGBKOsHOS1 and pGADOsICE1, respectively

Fig. 6 Bi-molecular fluorescence complementation assay (BiFC) to

test for interaction of OsHOS1 and OsICE1. The sequences of

OsHOS1 and OsICE1 were fused in frame with N-terminal (pYFN)

and C-terminal (pYFC) part of YFP, respectively. The two constructs

were used to transiently transform Arabidopsis protoplasts. As

positive control, two known interacting proteins were used. As

negative controls, pYFN-OsHOS1 and pYFC-OsICE1 were co-

transformed with pYFC43 and pYFN43 (Belda-Palazon et al. 2012)

empty vectors, respectively. BF bright field image, YFP Yellow

fluorescent protein image, Merge overlay of BF and YFP images.

Scale bar = 30 lm. On the right side of the panel, the bars represent

the average fluorescence signal intensity of 15 different protoplasts

for each transformation (error bars represent standard deviation). The

fluorescence signal intensity was analyzed using the MetaMorph�

software (Molecular Devices, USA). The asterisk (*) and double

asterisk (**) represent statistically significant differences (p \ 0.05,

Student’s t test analysis) between the different transformation

combinations used. Bars with the same mark (* or **) represent

absence of statistical difference between them

Plant Mol Biol

123

assess this, we followed the rate of degradation of a puri-

fied 6 9 HIS tagged OsICE1 protein (HIS-OsICE1) over

time, with or without addition of MG132 to crude plant

extracts. HIS-OsICE1 was rapidly degraded (in the first

15 min) when incubated with WT protein extracts while

the rate of degradation was slower with RNAi::OsHOS1

protein extracts (Fig. 7). The incubation of the protein

extracts with the proteasome inhibitor (MG132) slowed the

rate of degradation of HIS-OsICE1, showing the involve-

ment of a proteasome-dependent degradation pathway

(Fig. 7). Since the protein extracts were incubated with

MG132 at time 00 and not pre-incubated prior to the start of

the assay, we could also observe differential degradation of

the HIS-OsICE1 between the WT and transgenic protein

extracts (Fig. 7). These results showed that OsHOS1 is part

of the proteasomal complex and regulates the level of

OsICE1 by targeting it for degradation.

Discussion

Abiotic stress is a major constrain to crop productivity

worldwide. To tolerate these stresses in nature, plants have

evolved complex mechanisms to signal and respond

according to the intensity and duration of these stresses

(Saibo et al. 2009). Besides transcriptional changes, the

ubiquitin/26S proteasome (UPS) remodelling of the pro-

teome also plays an important role in the stress response

allowing plants to increase their chances of survival

(Dreher and Callis 2007).

In the present work, we were able to identify a rice

(O. sativa L., cv. Nipponbare) orthologue of the Arabid-

opsis HOS1 (AtHOS1), which was named OsHOS1 (Gen-

Bank accession no. JQ866627). This gene encodes a 942 aa

long protein with E3-ubq ligase activity, carrying a modi-

fied C3HC4 RING-finger motif similar to the AtHOS1 (Lee

et al. 2001). This RING-finger present in OsHOS1 is a

typical motif of several E3 ubiquitin ligases (Dreher and

Callis 2007; Joazeiro and Weissman 2000; Lyzenga and

Stone 2012).

The AtHOS1 has been reported as a negative regulator

of the cold stress response, mainly affecting DREB1A/

CBF3 expression (Ishitani et al. 1998; Lee et al. 2001). The

mechanism by which AtHOS1 negatively controls DREB1/

CBF expression involves the control of AtICE1 protein

level (Chinnusamy et al. 2003) through the ubiquitin/pro-

teasome pathway (Dong et al. 2006). Recently, AtHOS1

has also been involved in biological processes other than

cold stress signalling, such as flowering (Lazaro et al.

2012) and seed dormancy (Kendall et al. 2011). Previously,

it was reported that AtHOS1 was distributed throughout

the cytoplasm and nucleus (Lee et al. 2001). However,

recently, Tamura and co-workers proposed that AtHOS1

could be part of the nucleopore complex (Tamura et al.

2010). In rice, these biological functions of HOS1 are yet

to be confirmed and its interactome fully uncovered.

Another orthologue of AtHOS1, PtrHOS1, was identi-

fied in trifoliate orange [Poncirus trifoliata (L.) Raf.] (Liu

et al. 2010). PtrHOS1 is expressed at control temperatures

in different tissues but it is rapidly down-regulated (within

30 min), especially in leaves, upon cold stress (Liu et al.

2010). However, the PtrHOS1 transcript accumulation

returned to control levels by the end of the first hour of cold

stress. These results are in agreement with the previously

reported control of AtHOS1 expression by low temperature

(Lee et al. 2001). In our data set, the OsHOS1 transcript

level was mainly unaltered during cold stress as compared

to control conditions (Fig. 3a).

We have analyzed the role of OsHOS1 in the modulation

of cold stress response by generating an OsHOS1 silencing

rice line, as well as by transiently silencing OsHOS1 in rice

protoplasts. Although, in cold stress response we could

Fig. 7 Proteasome-mediated degradation of OsICE1. Crude protein

extracts from RNAi::OsHOS1 and WT plants under control condi-

tions were incubated at 30 �C with His-OsICE1, with or without

MG132 for different sampling time points. Ctrl represents the His-

OsICE1 input for all the assays without crude protein extract at time 0

or after 30 min of incubation at 30 �C. The different samples were

resolved on 10 % SDS-PAGE and the detection of HIS-OsICE1

was made as described in ‘‘Materials and methods’’ section. The

immunoblot detection of actin is shown as loading control

Plant Mol Biol

123

observe a higher expression of the OsDREB1A and a

DREB1/CBF target-gene (P5CS) in the silencing line as

compared to WT, no differences were observed regarding

plant survival or electrolyte leakage. A similar phenotype

was observed for the Arabidopsis KO mutant hos1 (Ishitani

et al. 1998). Despite having a higher expression of

DREB1A under cold, the plants failed to show increased

cold tolerance (Ishitani et al. 1998). These observations

indicate that HOS1 has a similar function in Arabidopsis

and rice and that the transient induction of DREB1A

observed in the transgenics is not enough to confer cold

stress tolerance. To improve cold stress tolerance we may

need to have a constitutive expression of DREB1A, as

observed in plants over-expressing DREB1A (Oh et al.

2005; Pellegrineschi et al. 2004).

We also analyzed the possible role of OsHOS1 in tar-

geting OsICE1 for degradation, as it could explain the

higher expression of OsDREB1A under cold stress in the

silencing line and in the transformed protoplasts. In our

experiments, OsICE1 showed a constitutive gene expres-

sion similar to that reported for Arabidopsis (Chinnusamy

et al. 2003) and rice (Nakamura et al. 2011). The OsICE1

expression was not altered by OsHOS1 silencing, sug-

gesting that OsHOS1 may act similarly to AtHOS1 by

targeting OsICE1 for degradation through the 26S protea-

some pathway. In fact, RNAi-OsHOS1 plants showed a

higher accumulation of OsICE1 as compared to WT

(Fig. 4). In addition, the incubation of rice plants with a

proteasome inhibitor led to an accumulation of OsICE1

under control conditions in both silencing line and WT,

thus confirming the role of the proteasome. We also

assessed the in vitro degradation rate of a recombinant

HIS-OsICE1 protein and found a faster degradation rate

with WT protein crude extracts as compared to extracts

from the RNAi line, probably due to the higher OsHOS1

accumulation in the WT. These results are in agreement

with previous data obtained from Arabidopsis (Dong et al.

2006). Interestingly, and opposite to what was previously

shown by Dong et al. (2006) for AtHOS1 and AtICE1, our

results showed that in Arabidopsis protoplasts in control

conditions, OsHOS1 can interact in vivo with OsICE1 in

the nucleus (Fig. 6). This result may indicate that either

OsHOS1 regulation of OsICE1 in rice is different from that

in Arabidopsis, or that the stress imposed to obtain pro-

toplasts is enough to promote the interaction between both

proteins.

Since ICE1 was shown to bind MYC-recognition cis-

elements in the promoters of DREB1/CBF genes (Chinnus-

amy et al. 2003), we used an in silico approach to search for

MYC recognition sites (putative targets of OsICE1) in the

promoter of OsDREB1A. Although some MYC recognition

sites are present in this promoter, they are much less abun-

dant than in the Arabidopsis DREB1A promoter

(Chinnusamy et al. 2003). It would be important to investi-

gate whether OsICE1 binds or not to the MYC recognition

sites present in the promoter of OsDREB1A and other Os-

DREB1 genes. Another possible function for ICE1 is the

repression of MYB15 transcripts (Miura et al. 2007). MYB15

is a known repressor of the DREB1/CBF regulon (Agarwal

et al. 2006) and it would be interesting to test the expression

pattern of a rice homologue of MYB15 in the RNAi::OsHOS1

plants in comparison to WT under cold stress.

Despite the higher OsICE1 protein level, the induction

of OsDREB1A gene expression in the transgenic plants was

transient in response to cold stress. The transient up-regu-

lation of DREB1/CBF and downstream target genes in

response to cold has been shown for several species

(Dubouzet et al. 2003; Gao et al. 2002; Gilmour et al. 1998;

Skinner et al. 2005; Wang et al. 2011) and is expected even

in the presence of higher ICE1 protein levels, as ICE1 is

known to be regulated by cold at post translational level.

The transient induction of OsDREB1A by cold was similar

between RNAi::OsHOS1 and WT, suggesting that OsICE1

must undergo post-translational modifications, such as

phosphorylation or SUMOylation to activate/stabilize the

protein for downstream target activation. It was shown that

two residues (K393 and S403) of AtICE1 could play a

major role in SUMOylation (Miura et al. 2007) and protein

stabilization and regulation (Miura et al. 2011) respec-

tively, affecting the ubiquitylation of ICE1. SUMOylation

of the K393 blocked polyubiquitination in vitro, but it is

not the primary target for ubiquitination (Miura et al.

2007). This reveals a competition for the same regions

between ubiquitination and SUMOylation thus regulating

protein stability and activity. A substitution in the S403

residue also blocked polyubiquination in vivo, but not

in vitro (Miura et al. 2011). We have also analyzed the

expression of OsSIZ1, known to have E3 SUMO activity

(Park et al. 2010), and no differences could be observed

between WT and the silencing line. It is possible that a

similar mechanism of competition between ubiquitination

and SUMOylation may intervene to regulate the stability/

activity of OsICE1 in rice under cold stress conditions.

All together, our results revealed that rice has also a

functional RING-finger E3 ubiquitin ligase (OsHOS1) with

function in the regulation of cold stress response similar to

the one found in Arabidopsis. OsHOS1 can physically

interact with OsICE1, modulating its abundance and down-

stream gene expression. However, as previously suggested

(Dong et al. 2006; Lee et al. 2001), we cannot exclude the

hypothesis that OsHOS1 acts outside the cold signalling

pathway or that OsICE1 may have different targets other

than the DREB1/CBF genes. Recently, it has been shown that

an ICE1 orthologue from banana (MaICE1) can interact with

MaMYC2, a component of the jasmonic acid signaling

pathway (Zhao et al. 2013). This interaction was correlated

Plant Mol Biol

123

with the chilling tolerance induced by MeJA in banana

(Zhao et al. 2013). Although under cold stress we could

observe a large resemblance between rice and Arabidopsis

in the regulation of OsICE1 by OsHOS1, we cannot discard

the hypothesis that targets of OsHOS1 are different from

those of AtHOS1. Therefore, it is of interest to unravel the

mechanism of action of OsHOS1 under other abiotic stresses

and at flowering stage, as well as to search for novel

interactors.

Acknowledgments This work was supported by the Fundacao para a

Ciencia e Tecnologia (FCT) through the projects POCI/BIA-BCM/

56063/2004, PTDC/BIA_BCM/099836/2008, Pest-OE/EBQ/LA0004/

2011. We would like to thank Dr. Ko Shimamoto (Nara Institute of

Science and Technology, Japan) for the Gateway-based vector,

pANDA, used in rice transformation for the production of the

RNAi::OsHOS1 plants and Dr. Alejandro Ferrando for providing

the BiFC vectors (www.ibmcp.upv.es/FerrandoLabVectors.php). TL

(SFRH/BPD/34943/2007), DDF (SFRH/BD/29258/2006), AC (SFRH/

BD/74946/2010) and IAA (SFRH/BPD/78314/2011) are grateful to

Fundacao para a Ciencia e a Tecnologia (FCT) for their fellowships. NS

was supported by Programa Ciencia 2007, financed by POPH (QREN).

Conflict of interest The authors declare that they have no conflict

of interest.

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