Supporting Information Legends
Fig. S1. (a) Set of peptides identified following tryptic peptide digestion and MS analysis of
a ~40 kDa protein band from nuclear fraction protein extract isolated from nuc mutants.
(b) Alignment of GLL23 sequence with its paralog GLL22 (At1g54000). The unique peptides
listed in (a) are highlighted in green. These specific amino acid sequences are present only in
At1g54010, whereas At1g54000 protein sequence does not match any of these peptide
sequences.
Fig. S2. (a) Sequence alignment (generated with ClustalW 2.1) of myrosinase-associated protein
GLL23 and the synthetic peptide used for the generation of the TUB-1A2 antibody (Kreis et al.,
1987). (b) Alignment of the same synthetic peptide sequence with Arabidopsis α-tubulin
sequence. Asterisks and dots under the sequence alignment indicate identical and similar
residues, respectively.
Fig. S3. Immunofluorescence labeling with TUB-1A2 of 10-d-old nuc, cyb and wild-type Col-0
seedlings expressing GLL23-RFP under pUBQ10prom. The arrowheads indicate nuclear cage
structures, whereas arrows point at representative cyb compartments. Yellow colour in the
merged images indicates co-localization. Note that GLL23-RFP labelled cyb and nuc
compartments are identical to TUB-1A2-labelled mutant structures. TUB-1A2 labels only
microtubules in wild-type seedlings. Scale bars = 10 µm.
Fig. S4. Sequence alignment of NUC (At1g54030) and closely related myrosinase-associated
proteins from Arabidopsis thaliana generated by Clustal Omega. Asterisks and dots under the
sequence alignment indicate identical and similar residues, respectively. The glycine residue
affected by the nuc mutation is highlighted in red. The sites of allelic mutations (mvp1-1 and
gold36) are highlighted in blue. The GDSL-lipase chain (as predicted by Pfam) is highlighted in
yellow. The residues forming the catalytic triad and serving as proton donors to the oxyanion
hole are highlighted in green.
Fig. S5. (a-c) Confocal images of the epidermal cells of 8-day-old wild-type Col-0 (a), cyb (b)
and nuc (c) seedlings transiently expressing either the Golgi marker Man1 (a, b) or ST-RFP (c).
1
(d-f) Confocal images of the epidermal cells of 8-day old wild-type Col-0 (d), cyb (e) and nuc (f)
seedlings transiently expressing a plasma membrane and apoplast marker YFP-LTPG.
The arrowheads indicate nuc compartments. Scale bars = 10 µm.
In contrast to partial retention of secretory markers to the ER (including aberrant perinuclear
structures) of nuc mutants, the cyb mutation does not affect the trafficking of the Golgi marker
Man1-mCherry and the plasma membrane and apoplast marker YFP-LTPG.
Fig. S6. Sequence alignment of CYB (At1g57620) and p24 proteins from Arabidopsis thaliana
generated by Clustal Omega. Asterisks and dots under the sequence alignment indicate identical
and similar residues, respectively. The glycine residue affected by the cyb mutation is boxed. The
GOLD domain of CYB (as predicted by ScanProsite software) is indicated by the blue line at the
top of the alignment.
Fig. S7. Immunoblot analysis with TUB-1A2 of cyb plants, wild-type plants expressing the
mutant (G96E) version of CYB under UBQ10prom (independent transgenic lines 2, 3, 8, 9, 10),
and cyb mutants expressing the wild-type CYB under UBQ10prom (independent transgenic lines 2
and 3). The arrowhead points at a ~40 kDa protein band recognized by TUB-1A2 in cyb mutants.
The 55kDa band corresponds to tubulin.
Table S1. (a) The genes co-expressed with GLL23/At1g54010. (b) Top 10 genes co-expressed
with GLL23 under various experimental perturbations (e.g. stress, chemical and hormone
treatments).
Table S2. Sequences of primers used for Gateway cloning.
Method S1. Positional cloning of CYB and NUC.
Method S2. RT-PCR
Video clip S1. The cyb compartments are highly motile. Time-lapse series of cyb compartments
visualized by GLL23-RFP construct in the cyb-1 mutant was acquired at 1 s intervals. Video
playback is at 1 frame per second (fps). Total elapsed time is 27 s. Bar = 5 μm.
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