Figure S1. Patterns of relative mean expression values in WT and wus mutant (accession number:
SAIL_150_G06) during LRP to SM transdifferentiation, corresponding to 9 clusters (of 18 generated by kmeans clustering) significantly enriched for different GO terms. GO category enrichment in clusters 1, 5,
6, 7, 10, 11 and 13 passed and FDR threshold of p<0.05 and the enriched terms are detailed in
supplementary table 13.
Figure S2. “Regeneration” eFP Browser, a bioinformatic tool for comparing gene expression patterns in
different in vitro regeneration systems. Example eFP output shown here represents RAP2.6L
(At5g13330), a gene previously shown to function in shoot regeneration from callus (Che et al 2006).
Highest expression levels (red) for this gene are revealed during shoot induction from root induced
callus and within sorted pWUS::mGFP5-ER expressing cells from transdifferentiating LRP.
Table S1. Over-represented Gene Ontology (GO) categories amongst genes differentially expressed in
wild type (Columbia-0) during cytokinin-induced LRP-SM conversion (19, 30 & 48 hours 2iP treatment).
Affymetrix ATH1 data processed with MAS5. Differentially expressed genes identified using limma
(linear models for microarrays) package (Smyth 2005), with a p-value cut-off of <0.05 and fold change
cut-off of >1.75.
Table S2. Expression of shoot meristematic genes (pale green fill "Gene" column) in the sampled
seedling primary roots is generally elevated after 2iP treatment, and expression of several root
meristem and LRP associated genes (light orange fill "Gene" column) is reduced. The PLETHORA genes
(PLT and BBM) are master regulators promoting basal/root fate, and all show reduced expression after
transfer to 2iP. Student’s t-test was performed on pair-wise comparisons between each of 2iP
treatment period points and 0 hours, significant p values <0.05 are highlighted (light blue fill). Expression
values for CLV3 and STM did not meet our absent call cut-off at any time point.
Table S3. Differentially expressed cytokinin signalling and cytokinin metabolic genes during LRP-SM
conversion. Many negative regulators of cytokinin (particularly type-A ARRs) and cytokinin catabolic
genes (cytokinin oxidases) showed increased expression on 2iP, consistent with mechanisms to maintain
cytokinin signaling homeostasis. Green fill marks increased mean expression values relative to the 0
hour time point >1.5 fold change, whereas red fill corresponds to a mean decrease in expression >1.5
fold. Significant p-values (<0.05) for pair-wise comparisons of each of the time points with 0 hours are
highlighted with light blue fill.
Table S4. Differentially expressed auxin-related genes during LRP-SM conversion. Green fill marks
increased mean expression values relative to the 0 hour time point >1.5 fold change, whereas red fill
corresponds to a mean decrease in expression values >1.5 fold. Significant p-values (<0.05) for pair-wise
comparisons of each of the time points with 0 hours are highlighted with light blue fill. Many auxin
signaling and metabolic genes are auxin-inducible and their expression levels were expected to fall after
the transfer from NAA to 2iP. Expression of the majority of indole-3-acetic acid inducible (IAA) and auxin
response factors (ARFs) conformed to this expectation with the exception of IAA3/SHY2, IAA16,
IAA17/AXR3, IAA28, IAA31 and ARF3/ETT which were up-regulated after transfer to cytokinin. These
observations broadly agree with a survey expression data from previous experiments (eFP browser refs
of exps too). Expression of IAA3, IAA16 & IAA17 have been shown to be modestly up-regulated by
exogenous cytokinin, whereas IAA28 transcription appears to be repressed by auxin and IAA31 is not
transcriptionally up-regulated by exogenous auxin. Treatment of seedlings with exogenous auxin or
cytokinin does not significantly affect the expression of ARF3, but cell specific expression studies
indicate high expression values for this gene in the rib meristem (Yadav et al.2009), and its increased
expression may reflect de novo induction shoot mertistematic tissues.
The PIN (PIN-FORMED) genes encode a family of auxin efflux carriers that mediate polar transport of the
hormone from cell to cell, regulating its localization. PIN1, PIN3 and PIN7 showed reduced expression
after transfer to 2IP. Interestingly, IAA3/SHY2, which showed increased expression on 2iP, is believed to
negatively regulate the PIN genes (Dello Ioio et al. 2008). IAA3 itself is activated by ARR12 (Dello Ioio et
al. 2008), which also showed increased expression on 2iP. The only PIN with increased expression on 2iP
was PIN6, which is normally localized to specific regions of developing LRPs, but becomes more broadly
expressed after exogenous cytokinin treatment (Laplaze et al. 2007). It has been hypothesized that this
change in PIN6 expression may play an important part in the inhibition of LRP by cytokinin, by obscuring
organ boundaries (Laplaze et al.2007).
Table S5. Cell cycle-related DEGs identified in transcriptomic analysis of cytokinin-induced LRP-SM
conversion. Many positive regulators of the cell-cycle show reduced expression after transfer to 2iP,
whereas expression of some negative regulators (e.g. KRP1 & KRP2) is increased. Green fill marks
increased mean expression values relative to the 0 hour time point >1.5 fold change, whereas red fill
corresponds to a mean decrease in expression values >1.5 fold. Significant p-values (<0.05) for pair-wise
comparisons of each of the time points with 0 hours are highlighted with light blue fill. Cytokinins are
generally regarded as essential in promoting cell division in plants, but have been shown to inhibit LRP
initiation, apparently through down-regulating genes involved in the G2 to M transition (Mironov et al.
1999, Himanen et al. 2002, De Veylder et al. 2003, Li et al. 2006). Cytokinin can also decrease the
number of dividing cells and the size of the RAM (Beemster and Baskin 2000, Werner et al. 2003, Li et al
2006). LRP induction certainly involves strong synchronous up-regulation of cell division in our samples.
Transfer to high concentrations of cytokinin might limit immediate cell division subsequently as it
promotes fate changes/differentiation in a proportion of the formally dividing cells. Cell division is
dependent upon the accumulation of the resources necessary for subsequent genome duplication and
division. Up-regulation of photosynthetic machinery and chloroplast division might temporarily limit
resources for cell division. With regard to increased expression of cyclin-dependent kinase inhibitors,
KRP2 has been identified as a negative regulator of LRP formation (Sanz et al. 2011) and it is interesting
that it is up-regulated during the transition from LRP to SM. KRP1 and KRP2 are believed to promote
endoreduplication and increased cell size, which may be associated with differentiation and the
assumption of specific fates. Four cyclins are up-regulated after the transfer to 2iP, CYCD6;1, CYCP3;2,
CYCP4;2 and CYCP4;3. Type P cyclins are as yet a poorly characterized group, but have been
hypothesized to offer a potential link between nutritional status and regulation of the cell cycle (Acosta
et al. 2004). It is interesting that CYCD6;1 expression was increased by the cytokinin treatment. CYCD6;1
has been shown to be positively regulated by SHR and SCR in controlling divisions that give rise to root
ground tissues (Sozzani et al. 2010), but SHR and SCR are both down-regulated by 2iP, suggesting
another mechanism is responsible for up-regulating CYCD6;1. Histone and Chromatin-related DEGs
generally show reduced expression after transfer to 2iP. Reduced expression of many of these targets
likely reflects the impact of cytokinin in negatively regulating the cell cycle in transforming LRP.
Table S6. Common targets identified in transcriptome analyses by Che et al. (2006) (regeneration from
root-derived callus) and our study of cytokinin-induced LRP to SM conversion. Many targets related to
shoot organogenesis are similarly regulated during LRP-SM conversion. Green fill marks increased mean
expression values relative to the 0 hour time point >1.5 fold change, whereas red fill corresponds to a
mean decrease in expression values >1.5 fold. Significant p-values (<0.05) for pair-wise comparisons of
each of the time points with 0 hours are highlighted with light blue fill.
Table S7. Expression of genes previously identified as modulating in vitro shoot regeneration during LRPSM conversion. Green fill marks increased mean expression values relative to the 0 hour time point >1.5
fold change, whereas red fill corresponds to a mean decrease in expression values greater than 1.5 fold.
Significant p-values (<0.05) for pair-wise comparisons of each of the time points with 0 hours are
highlighted with light blue fill.
Table S8. Genes differentially expressed in a wus mutant (line accession: SAIL_150_G06) during
cytokinin-induced LRP to SM transdifferentiation.
Table S9. Over-represented GO terms amongst DEGs identified in transcriptome comparison between
homozygous wus (SAIL_150_G06) mutant and wild type during cytokinin-induced LRP to SM conversion.
GO analysis performed using the AGRIGO analysis toolkit and database
(http://bioinfo.cau.edu.cn/agriGO/). Singular enrichment analysis was used with Fisher as the statistical
test method.
Table S10. Significantly enriched GO terms corresponding to 7 clusters (of 18 generated by k-means
clustering) derived from genes differentially expressed in a wus loss-of-function background (accession
number: SAIL_150_G06) during LRP to SM conversion. GO analysis was performed using the AGRIGO
analysis toolkit and database (http://bioinfo.cau.edu.cn/agriGO/). Singular enrichment analysis was used
with Fisher as the statistical test method, Hochberg false-discovery rate method, and a minimum
significance level of p<0.05.
Table S11. Genes differentially expressed in the root of two wus alleles (line accessions: SAIL_150_G06
and GABI-KAT_870H12 ) compared with wild type during cytokinin-induced LRP to SAM conversion, after
30 hours treatment with 2iP.
Table S12. Candidate homozygous TDNA insert lines tested (2 or more replicates) for differences in LRPSAM conversion and found not to differ significantly from wild type.
Table S13. Pearson correlation coefficients for comparisons between arrays used in cell-specific profiling
studies. High values (close to 1) were obtained for comparisons between our sorted pWUS::GFP-ER cells
from tLRP after 30 hours 2iP treatment, suggesting a high degree of reproducibility in our sampling. Low
coefficients in comparisons between these cells and our whole tissue samples likely reflect significant
differences between the domain sampled and the whole sample, compounded by the partial induction
of shoot meristematic cells in root tissue. The transcriptome of pWUS::GFP-ER cells from converting LRP
also differed very significantly from those sampled from apetala1/cauliflower double mutant SAMs
(WUSp, Yadav et al. 2009), which likely reflects differences in the organs sampled and the high
concentrations of hormones used to induce LRP to SM conversion.
Table S14. Genes differentially expressed in FACS sorted cells expressing pWUS::mGFP-ER from LRP
undergoing conversion to SMs (30 hours 2iP) compared with the WUSp expression domain of
apetala1/cauliflower double mutant SAMs isolated (Yadav et al. 2009). Shown are 200 significantly
differentially expressed genes (p<0.05) with the greatest fold difference in expression between WUSp
cells from tLRP (30 hours 2iP) and from SAMs.
Table S15. Enriched GO terms amongst DEGs identified in the transcriptome of FACS sorted cells
expressing pWUS::mGFP-ER from LRP undergoing conversion to SMs (30hours 2iP) compared with the
WUSp expression domain of apetala1/cauliflower double mutant SAMs (Yadav et al. 2009). GO analysis
was performed using the AGRIGO analysis toolkit and database (http://bioinfo.cau.edu.cn/agriGO/)
Singular enrichment analysis was used with Fisher as the statistical test method, Yekutieli multi-test
adjustment.
Methods S1. Quantitative Real-Time PCR (qRT-PCR) validation of ATH1 arrays.