Figure S1. Selection of T-DNA lines. (a) Position of T-DNA insertions in RPX (At5g04410)
coding region as used in this study. (b) Expression profile of RPX during leaf expansion as
adapted from Beemster et al. (2005). (c) Photograph showing cleared leaves of the first leaf
pair of wild-type (wt) and the second knock-out line (rpx-2) after 22 days of growth. (d) From
left to right: leaf area, mesophyll cell size and cell number of wild-type and rpx-2 plants. For
each data point, n = 20, with 50-100 measurements per leaf; means  SE. (e) Phenotypes of
wild-type (wt), rpx-1 mutant, 35S::RPX (ox) and 35S::RPX∆C (oxc) plants. Plants were
grown in soil at 21°C under long-day condition for 5 weeks.
Figure S2. Analysis of the hydrophobic C-terminus of the RPX protein and phenotype of the
transgenic 35S::RPX∆C plants. (a) Hydropathy plot of the amino acid sequence of RPX as
determined by Protscale (http://web.expasy.org/cgi-bin/protscale/protscale.pl). (b) Protein
sequence alignment of Arabidopsis thaliana RPX (At5g04410) and its close homologue
ANAC053 (At3g10500), their orthologues from Arabidopsis lyrata (939838_78 for RPX, and
478384_53 for ANAC053); the constructed truncated RPX∆C sequence, which lacks the
highly conserved C-terminal region, is shown as well. (c) Pleiotropic phenotypic effects
caused by the overexpression of RPX∆C in Arabidopsis. Phenotypes shown are stunted
growth and early flowering, the appearance of an aerial rosette and the twisting and bending
of rosette leaves. Plants were grown in soil at 21°C under long-day condition for 5 weeks.
Figure S3. Subcellular localization and tissue-specific expression pattern of RPX. (a - b)
Both, full-length and truncated RPX proteins (fused to green fluorescent protein) are
localized in the nucleus although some fluorescent signal was also observed within the
cytoplasm. GUS activity driven by the RPX promoter was detected mainly in the vascular
tissue of full-grown leaves (c), roots (d), stem (e), and carpels (f). Cross section of a leaf
confirming that GUS activity is mainly within the vascular cells including the surrounding
bundle sheath cells (g). Transformation of tobacco (Nicotiana tabacum) with the RPX
promoter - GUS construct results in a leaf expression pattern similar to that in Arabidopsis
(h).
Figure S4. Inducible overexpression of RPX. (a) The inducible RPX∆C construct used for the
identification of target genes. (b) Determination of the optimal duration of induction by qRTPCR. (c) GO slim annotation (http://www.geneontology.org/GO.slims.shtml) and percentage
distribution of the 80 genes differentially expressed upon induction of RPX for 2 h.
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Figure S5. MapMan analysis of microarray data. MapMan (http://mapman.gabipd.org/)
analysis of the transcriptomics data obtained after 2 h of RPX induction revealed a strong
activation (red) of the expression of genes encoding subunits of the 26S proteasome.
Figure S6. Abiotic stress induction of RPX. The induction of RPX expression after 1 h and 3
h of UV-B stress is followed by an increase in the expression of genes encoding proteasomal
subunits. Moreover, after 12 h of UV-B stress RPX is no longer induced and the expression
of the 26S proteasomal genes drops to the basic levels again (data extracted from Kilian et
al., 2007).
Figure S7. Evolutionary conservation of the RPX binding site. Location of the identified
PRCE element (indicated by red boxes) in the promoters of genes encoding the 26S
proteasome in (a) Arabidopsis thaliana, (b) Oryza sativa, (c) Ricinus communis, and (d)
Chlamydomonas reinhardtii. Numbers after the species names (in brackets) indicate the
number of promoters containing the PRCE element relative to all promoters analyzed.
Numbers at the bottom of each panel indicate distance from the transcription start site
(located at position ´0´). Data plotted were obtained as described in Experimental
procedures.
Figure S8. Binding of RPX in vitro to the PRCE element. (a) Location of sequences used for
IMAP probes based on the RPN10 promoter. Numbers at the bottom indicate distance from
the transcription start site (located at position ´0´). (b) EMSA with HIS-tagged in vivo
expressed RPX∆C protein. Incubation of infrared (IR)-labeled oligo 1 containing the PRCE
element results in a retention of RPX (indicated by the arrow), which disappears upon
competition with a non-labeled oligo 1.
Figure S9. Position of primers used for ChIP-qPCR. Grey, promoter; red, 5’ and 3’ UTR;
orange, exon; blue, intron; green, PRCE element.
Figure S10. Proteasome abundance and activity. (a) Immunological detection of PBE1 in
leaves of RPX transgenics and wild-type (wt) plants. As loading control, the abundance of
the large subunit of Rubisco is shown. (b) Relative 20S proteasome activity after 8 hours of
estradiol treatment in a RPX∆C-IOX plant. 20S proteasome activity was determined for five
independent biological replicates during 2 hours of incubation. Fluorescence was measured
at 5-min intervals in a plate reader (Ex. 360 nm, Em. 460 nm). Subsequently, individual
measurements were plotted and the slope for each sample was calculated. The % activity of
control was determined as follows: estradiol treatment slope / control treatment slope x 100.
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The asterisk indicates significant difference in proteasome activity (P < 0.05). (c) Overall
proteasome activity in RPX transgenics as compared to wild type. Quantification of
proteasome-derived signals in Arabidopsis leaves. Fluorescently labeled proteins were
detected on protein gels by fluorescence scanning. The intensity of the overall proteasomal
bands was quantified by image analysis software (Scion Image 4.0.3.2) and normalized
based on the level of the large subunit of Rubisco. Data represent means ± SD. Two-sample
tests between wt overall proteasomal band intensity and the remaining samples were done
(P < 0.05, non-parametric Mann-Whitney U test).
Table S1. List of genes that are differentially expressed after induction of RPX for 2 hours.
Table S2. Primer sequences.
Table S3. Expression analysis of genes encoding for the 26S proteasome.
Methods S1. Infrared-mediated mapping (IMAP).
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