SUPPLEMENTAL DATA LEGENDS
Figure S1: Stages to produce the constructs used in this work (see Table 1).
Figure S2: Aberrant growth phenotype of several homozygous AtBT1::T-DNA mutants.
(a) General overview of 2-weeks old WT and AtBT1::T-DNA plants cultured in solid
MS supplemented with 90 mM sucrose. (b) Different phenotypes displayed by
AtBT1::T-DNA mutants cultured in solid MS supplemented with sucrose.
Figure S3: Mitochondrial delivery of AtBT1 complements the aberrant growth and
sterility phenotype of homozygous AtBT1::T-DNA mutants. (a) PCR analysis of
genomic DNA from WT plants and from homozygous AtBT1::T-DNA plants
complemented with TP´´-AtBT1 (COM), using the LP and RP AtBT1-specific primers,
and the T-DNA left border-specific LBb1 primer (see Experimental Procedures, cf.
Figure 1a). (b) PCR analysis of genomic DNA from WT plants and homozygous
AtBT1::T-DNA plants complemented with TP´´-AtBT1 using specific primers for 35S
promoter and for the AtBT1 3´ end (see Experimental Procedures and Figure 1c). (c)
PCR analysis of genomic DNA from WT plants and homozygous AtBT1::T-DNA plants
complemented with TP´´-AtBT1 using specific primers for the sequence that codes for
the 17 amino acids at the N-terminal extension of AtBT1 and for the AtBT1 3´ end (see
Experimental Procedures). (d) Growth pattern of 2-months old WT plants and
homozygous
AtBT1::T-DNA
mutants
complemented
with
TP´´-AtBT1.
(e)
Morphology of seeds from WT plants and from the first generation of homozygous
AtBT1::T-DNA mutants complemented with TP´´-AtBT1. In “b”, note that specific
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primers for 35S and AtBT1 3´ end can amplify a ca. 1400 bp PCR fragment in TP´´AtBT1 expressing AtBT1::T-DNA plants, but not in WT plants. In “c”, note that specific
primers for the sequence that codes for the 17 amino acids at the N-terminal extension
of AtBT1, and for the AtBT1 3´ end can amplify a ca. 1500 bp PCR fragment in WT
plants, but not in TP´´-AtBT1 expressing AtBT1::T-DNA plants.
Figure S4: Mitochondrial delivery of AtBT1 complements the aberrant growth and
sterility phenotype of homozygous AtBT1::T-DNA mutants. (a) PCR analysis of
genomic DNA from WT plants and from homozygous AtBT1::T-DNA plants
complemented with AtBT1pro-TP´-AtBT1 (COM), using the LP and RP AtBT1specific primers, and the T-DNA left border-specific LBb1 primer (see Experimental
Procedures, cf. Figure 1a). (b) PCR analysis of genomic DNA from WT plants and
homozygous AtBT1::T-DNA plants complemented with AtBT1pro-TP´-AtBT1 using
specific primers for AtBT1pro and for the 35S terminator (see Experimental
Procedures). (c) PCR analysis of genomic DNA from WT plants and homozygous
AtBT1::T-DNA plants complemented with AtBT1pro-TP´-AtBT1 using specific
primers for the sequence that codes for the 8 amino acids at the N-terminal extension of
AtBT1 and for the AtBT1 3´ end (see Experimental Procedures). (d) Growth pattern of
2-months old WT plants and homozygous AtBT1::T-DNA mutants complemented with
AtBT1pro-TP´-AtBT1. (e) Morphology of seeds from WT plants and from the first
generation of homozygous AtBT1::T-DNA mutants complemented with AtBT1pro-
TP´-AtBT1. In “b”, note that specific primers for AtBT1pro and AtBT1 3´ end can
amplify a ca. 2500 bp PCR fragment in AtBT1pro-TP´-AtBT1 expressing AtBT1::TDNA plants, but not in WT plants. In “c”, note that specific primers for the sequence
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that codes for the 8 amino acids at the N-terminal extension of AtBT1, and for the
AtBT1 3´ end can amplify a ca. 1500 bp PCR fragment in WT plants, but not in
AtBT1pro-TP´-AtBT1 expressing AtBT1::T-DNA plants.
Figure S5: TP´-AtBT1 is exclusively localized in mitochondria. General overview of
GFP fluorescence and red chlorophyll autofluorescence localization in leaf areas
including trichome, epidermis and mesophyll cells of (a) TP´-AtBT1-GFP-, (b) AtBT1GFP- and (c) MitTPr-GFP- expressing Arabidopsis plants. The arrows indicate
mitochondria
(m),
seen
as
small
punctuate
green
structures,
chlorophyll
lacking/deficient plastids of epidermal cells (p), and chlorophyll containing chloroplasts
(chl). Inset in “b” highlights the localization of AtBT1-GFP in epidermal chlorophyll
lacking/deficient plastids having labeled long stroma-filled tubular extensions
corresponding to plastid stromules. No such labeling was observed in TP´-AtBT1GFP- and MitTPr-GFP- expressing Arabidopsis plants (“a” and “c”, respectively). Note
that GFP fluorescence distribution in TP´-AtBT1-GFP expressing plants was identical
to that of the mitochondrial marker in MitTPr-GFP expressing plants, which is further
supported by movies shown in Figures S6 and S8. Bar = 20 m.
Movie S1: Movie showing distribution pattern, size, shape and high motility of GFP
fluorescence in TP´-AtBT1-GFP expressing Arabidopsis leaves.
Movie S2: Movie showing distribution pattern, size, shape and motility of GFP
fluorescence in AtBT1-GFP expressing Arabidopsis leaves.
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Movie S3: Movie showing distribution pattern, size, shape and high motility of GFP
fluorescence in MitTPr-GFP expressing Arabidopsis leaves.
Table S1: Primers used for the production of constructs used in this study.
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