Methods S1. Experimental procedures for all supplementary figures and tables.
Plasmid construction and plant transformation
To generate the GUS reporter gene driven by the RhHB1 promoter (GenBank
accession no. KF729512), a 1,650-bp fragment upstream of RhHB1 ATG was PCR
amplified with the primers RhHB1pF1 (containing a HindIII site at the 5' end) and
RhHB1pR1 (containing a BamHI site at the 3' end) and cloned into the binary vector,
pBI121, to replace the 35S CaMV promoter. For construction of the PRhGA20ox1:GUS
vector, a 1,427-bp fragment (GenBank accession no. KF729514) upstream of the
RhGA20ox1 ATG was PCR amplified using the primers RhGApF2 and RhGApR2
and inserted into the binary pBI121 vector to replace the 35S CaMV promoter.
Mutation of the 9-bp core-binding motif, AATATTATT, in the RhGA20ox1 promoter
was generated using PCR-based site-directed mutagenesis with the primers
RhGApmF and RhGApmR, and was designated as PRhGA20ox1m and transferred into the
pBI121 vector to generate PRhGA20ox1m:GUS.
To generate the RhHB1 overexpression binary vector, The ORF of RhHB1 was
amplified using the primers RhHB1F3 and RhHB1F3. The resulting fragment was
inserted into a modified binary pCAMBIA 1300 vector harboring a super promoter
and a GFP (Gong et al., 2002). The resulting constructs were transformed into
Agrobacterium strain GV3101 and then introduced into Arabidopsis plants using the
floral dip method (Clough and Bent, 1998).
Soluble protein content and ion leakage quantification
Soluble protein and relative electrolyte leakage were determined according to the
methods reported by Trivellini et al. (2012). In brief, 100 mg of material was ground
to a fine powder in liquid N2 and added to 500 μl of extraction buffer (100 mM TrisHCl, pH 7.6, 10 mM MgSO4, and 10 mM dithiothreitol). The samples were incubated
on ice for 1 h with intermittent vortexing, and the cellular debris was pelleted by
centrifugation at 10,000g for 10 min at 4°C. Soluble protein concentration in the
supernatant was then determined using the Coomassie dye-binding assay (Bio-Rad
Laboratories, Richmond, CA, USA) with bovine serum albumin as the standard.
To determine relative electrolyte leakage, tissues at each time point were placed
in a 50-ml tube containing 20 ml of deionized water and incubated at 25°C for 30 min
on an orbital shaker (200 rpm). The initial conductivity of the fluid was measured
with a conductivity detector (Shanghai INESA, Shanghai, China). The tissues were
then boiled for 10 min in deionized water and cooled to room temperature. The total
conductivity was then determined as before, and the relative electrolyte leakage was
expressed as the percentage of the initial conductivity versus total conductivity.
Transient expression assay
The Agrobacterium strain GV3101 transformed with the PRhHB1:GUS construct
was cultured for 24 h in 10 ml of LB medium containing 10 mM MES, 20 mM
acetosyringone, 50 μg ml-1 kanamycin and 50 μg ml-1 gentamycin sulfate. The
cultures were centrifuged, and the pelleted bacteria were resuspended in an
inoculation buffer (10 mM MgCl2, 200 mM acetosyringone, and 10 mM MES, pH
5.6). Rose (R. hybrida ‘Honey’) petals were injected with the Agrobacterium
suspension using needle-less syringes. For phytohormone treatments, the injected
samples were treated with 100 μM ABA or 10 μl L-1 ethylene for 24 h, as previously
described and then transferred to 22°C for 3 d in the dark.
The Arabidopsis mesophyll protoplasts were isolated as described in Yoo et al.
(2007). The plasmids were extracted from transformed E. coli DH5α cells using the
MACHEREY-NAGEL nucleic acid purification kit (MACHEREY-NAGEL, Düren,
Germany), then 10 μg effector plasmid and 10 μg reporter plasmid were transformed
into 100 μl protoplasts containing ~2×104 protoplasts by polyethylene glycol
mediated transformation.
Histochemical staining and quantitative assay of GUS activity
GUS activity and staining assays were carried out as described by Jefferson et al.
(1987). For staining, tissues were submerged in a staining solution containing 0.5 mg
ml-1 X-Gluc in 0.1 M Na2HPO4, pH 7.0, 10 mM Na2EDTA, 0.5 mM potassium
ferricyanide/ferrocyanide, and 0.06% Triton X-100. Samples were infiltrated under
vacuum for 5 min and then incubated at 37°C overnight before visualization. The
staining buffer was removed, and the samples were cleared in 70% ethanol. All
imaging was performed using a Nikon digital camera.
Accession Numbers
Sequence data from this article can be found in the Arabidopsis Genome Initiative or
GenBank/EMBL databases under the following accession numbers: RhHB1
(KF729511),
RhGA20ox1
(KF729513),
RhUBI1
(JK622648),
ACTIN2
(NM_112764), AtHB1 (AT3G01470), AtHB3 (AT5G15150), AtHB5 (AT5G65310),
AtHB6 (AT2G22430), AtHB7 (AT2G46680), AtHB12 (AT3G61890), AtHB13
(AT1G69780),
AtHB16
(AT4G40060),
AtHB20
(AT3G01220),
AtHB21
(AT2G18550),
AtHB22
(AT2G36610),
AtHB23
(AT1G26960),
AtHB40
(AT4G36740),
AtHB51
(AT5G03790),
AtHB52
(AT5G53980),
AtHB53
(AT5G66700), AtHB54 (AT1G27045), LeHB1 (AM902288), HaHB4 (AF339748),
MtHB1 (XP_003627511), GmHB (ACU24436), NbHB1 (ADH04264), PtHB
(XP_002302659),
(NP_001183573),
SbHB
(XP_002462710),
AtGA20ox1
VvHB
(AT4G25420),
(XP_002262950),
AtGA20ox2
ZmHB
(AT5G51810),
AtGA20ox3 (AT5G07200), AtGA20ox4 (AT1G60980), AtGA20ox5 (AT1G44090).
SUPPORTING REFERENCES
Clough, S.J. and Bent, A.F. (1998) Floral dip: a simplified method for
Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16,
735–743.
Gong, Z., Lee, H., Xiong, L., Jagendorf, A., Stevenson, B., and Zhu, J.K. (2002)
RNA helicase-like protein as an early regulator of transcription factors for plant
chilling and freezing tolerance. Proc. Natl Acad. Sci. USA. 99, 11507–11512.
Jefferson, R.A. (1987) Assaying chimeric genes in plants: the GUS gene fusion
system. Plant Mol. Biol. Rep. 5, 387–405.
Trivellini, A., Jibran, R., Watson, L.M., O’Donoghue, E.M., Ferrante, A.,
Sullivan, K.L., Dijkwel, P.P. and Hunter D.A. (2012) Carbon deprivationdriven transcriptome reprogramming in detached developmentally arresting
Arabidopsis inflorescences. Plant Physiol. 160, 1357–1372.
Yoo, S.D., Cho, Y. and Sheen, J. (2007) Arabidopsis mesophyll protoplasts: a
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1572.