Supporting Information - Legends
Figures
Figure S1. Biomass accumulation in dgt and VFN8 reciprocal heterografted plants. Plants
assayed are those used for the experiments shown in Fig. 1. Fresh weights of shoots (a) and roots
(b) were measured 4 weeks after grafting. (c) Shoot-to-root ratio, on a fresh weight basis, in dgt
and VFN8 reciprocal heterografted tomato plants.
Figure S2. Dynamics of the change in xylem differentiation in stems of dgt rootstocks grafted to
control (VFN8) scions. (a-c) Anatomical features of stem vascular bundles from dgt rootstocks
grafted to dgt scions; stem tissues were excised 10 (a), 20 (B) and 30 (c) days after grafting. (d-f)
Anatomical features of stem vascular bundles from VFN8 rootstocks grafted to VFN8 scions;
stem tissues were excised 10 (d), 20 (e) and 30 (f) days after grafting. (g-i) Anatomical features
of stem vascular bundles from dgt rootstocks grafted to VFN8 scions; stem tissues were excised
10 (g), 20 (h) and 30 (i) days after grafting. Scale bar: (a) = 100 µm, common to (b-i).
Figure S3. Dynamics of the change in stem anatomy of dgt rootstocks grafted to control (VFN8)
scions. (a-c) Anatomical features of stems from dgt rootstocks grafted to dgt scions; stem tissues
were excised 10 (a), 20 (b) and 30 (c) days after grafting. (d-f) Anatomical features of stem
vascular bundles from VFN8 rootstocks grafted to VFN8 scions; stem tissues were excised 10
(d), 20 (e) and 30 (f) days after grafting. (g-i) Anatomical features of stem vascular bundles from
dgt rootstocks grafted to VFN8 scions; stem tissues were excised 10 (g), 20 (h) and 30 (i) days
after grafting. Higher magnification of the vascular bundle (marked with dashed lines) is located
in the bottom left side of each image. Scale bar: (a) = 1000 µm, common to (b-i).
Figure S4. SlCyp1 transcripts are not phloem-mobile. Grafting experiments were performed with
dgt mutant and transgenic tomato (pSuc2:6xHis-SlCyp1) (TG) plant lines. RNA samples
extracted from the indicated rootstock stems (RS-Stem) and roots (RS-Root) were analyzed by
RT-PCR using primers specific for 6xHis-SlCyp1 and tubulin (as an internal control).
Figure S5. Expression of SlCyp1 in roots as compared to source leaves. (a) Relative
transcription level of SlCyp1 in source leaf and young developing roots of 4-week-old VFN8
plants. Transcription levels were normalized using tubulin as an internal control. The indicated
data represents means of 4 biological replications ± SE. Different letters indicate significant
differences at p < 0.05 by Student’s independent t-test. (b) Western blot analysis of proteins
extracted from root and source leaf of tomato plants. SlCyp1 detection was performed using
40µg of total protein.
Figure S6. Effect of light intensity on long-distance movement of SlCyp1 and the shoot-to-root
ratio. Light intensity experiments were conducted in a growth chamber. (a) Quantitative analysis
of data obtained from three Western blot experiments on proteins from stems of VFN8 and dgt
rootstocks grafted onto transgenic control scions expressing GFP under the AtSuc2 promoter
(pSuc2:GFP). Plants were exposed to PPFD of 120 and 280 µmole m-2 s-1. Values are presented
as the ratio between the integrated optical densities (IOD) of SlCyp1 relative to GFP. Analysis
was performed using Gelpro3 software. (b) Effect of light intensity on shoot-to-root ratio in
reciprocal dgt and VFN8 grafted plants. Note that significant response to PPFD was observed
only when VFN8 (expressing SlCyp1) served as the scion. Absence of SlCyp1 from the scion
alleviated the effect of PPFD on shoot-to-root ratio.
Tables
Table S1. Summary of enrichment testing within functional gene sets (gene set) of S.
lycopersicum genes differentially expressed between two graft combinations (gt1 and gt2) at
FDR<0.05 and FC>2 and mean expression count>2. Single-sided Fisher exact tests done on
the contingency tables (ratio) shown as “x/y to w/z”, z=number of assayed genes in tomato
genome, w=number of genes in the functional gene set, y=number of D.E. genes, and x=D.E.
genes that are members of the gene set; up1~up2 is number of genes higher in graft gt1 or in
gt2; Fisher test p-values: gtr.pvalue/stars (over-representation) and less.pvalue/stars (underrepresentation), *** for p<0.001, ** for p<0.01, * for p<0.05.
Table S2. Genes in lateral root initiation Cluster 5 functional gene set that were significantly
over-represented in the two graft comparisons dgt/dgt vs VFN8/VFN8 (13 genes) and dgt/dgt vs
VFN8/dgt (9 genes). slgene: S. lycopersicum gene ID; FC: fold change between gt1 and gt2;
atgene: A. thaliana gene ID of which slgene is putative ortholog; four graft-pair columns: mean
normalized expression in root RNA-seq experiment. symbols and desc from TAIRi.
Table S3. Genes in Auxin Response Factor (ARF) functional gene set that were
significantly over-represented in the two graft comparisons dgt/dgt vs VFN8/VFN8 (6
genes) and dgt/dgt vs VFN8/dgt (9 genes). slgene: S. lycopersicum gene ID; FC: fold
change between gt1 and gt2; atgene: A. thaliana gene ID of which slgene is putative
ortholog; four graft-pair columns: mean normalized expression in root RNA-seq
experiment. symbols and desc from TAIRi.
Table S4. Primer pairs used in the current study.
Methods
Methods S1. Gene ontology and gene set enrichment analyses.
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Datasets
Data S1. Differentially expressed genes. Each worksheet contains a list of the differentially
expressed genes in the following grafting comparisons: dgt/dgt Vs. VFN8/VFN8, dgt/dgt Vs.
VFN8/dgt and VFN8/dgt Vs. VFN8/VFN8. Column titles are as follows: Gene: The differentially
expressed gene ID. VFN8/VFN8, VFN8/dgt and dgt/dgt: Normalized expression values for each
of the indicated grafting. Fold change: The fold change for the specified gene between the
compared graftings. Adjusted p-val: The adjusted p-value for the gene differential expression.
Symbols: The gene symbols. Description: Gene annotation.
Data S2. Rescue of dgt root gene expression when grafted to VFN8 scions. List of genes
expressed in dgt roots grafted to VFN8 scions, whose expression level was different from that of
homografted dgt roots and similar to homografted VFN8 roots. Column titles are as follows:
Gene: The gene ID. VFN8/VFN8, VFN8/dgt and dgt/dgt: Normalized expression values for each
of the indicated grafting. Symbols: The gene symbols. Description: Gene annotation.
Data S3. Rescue of dgt root transcription factor expression when grafted to VFN8 scions. List of
transcription factors expressed in dgt roots grafted to VFN8 scions, whose expression level was
different from that of homografted dgt roots and similar to homografted VFN8 roots. Column
titles are as follows: Gene: The gene ID. VFN8/VFN8, VFN8/dgt and dgt/dgt: Normalized
expression values for each of the indicated grafting. Symbols: The gene symbols. Description:
Gene annotation.
Data S4. Rescue of dgt NAC domain gene expression when grafted to VFN8 scions. List of
NAC domain genes expressed in dgt roots grafted to VFN8 scions, whose expression level was
different from that of homografted dgt roots and similar to homografted VFN8 roots. Column
titles are as follows: Gene: The gene ID. VFN8/VFN8, VFN8/dgt and dgt/dgt: Normalized
expression values for each of the indicated grafting. Symbols: The gene symbols. Description:
Gene annotation.
Data S5. Lists of genes in the four functional gene sets LRI, DevXylem, MtrXylem, and ARF. Each
list includes column atgene containing the A. thaliana gene ID and column slgene containing the S.
lycopersicum gene ID of an OrthoMCL ortholog. (See the Attached Excel Spreadsheet.)
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Lamesch, Philippe, et al. "The Arabidopsis Information Resource (TAIR): improved gene
annotation and new tools." Nucleic acids research, 40.D1 (2012): D1202-D1210.
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