FIGURE LEGENDS FOR SUPPORTING INFORMATION
Supporting Table 1. PIN family gene expression in Arabidopsis nectaries and reference
tissues. Normalized mean ATH1 GeneChip probe set signal intensity for Arabidopsis PIN6
expressed in nectaries. Original data for all tissues was described in Kram BW et al. (2009)
BMC Plant Biol., 9:92. ILN = immature lateral nectaries, MLN = mature lateral nectaries, MMN
= mature median nectaries. Mean value derived from an n of 3 for all tissues except MMN (n =
2).
Supporting Table 2. RNAseq count data for pin6-2 and myb57-2 mature lateral nectaries.
The pin6-2 and myb57-2 mutants phenocopy each other in having smaller lateral nectaries,
reduced nectar production, and missing short stamen. Illumina-based RNA-seq was performed
on RNA isolated from the mature lateral nectaries of wild-type, pin6-2 and myb57-2 plants. This
study was performed to identify genes with altered expression in these mutants. The first tab
contains counts for all genes expressed in nectary tissues. The second and third tabs contain lists
of genes with counts 2-fold or higher, or lower, in Col-0 over myb57-2 and pin6-2. Other tabs
contain lists of genes differentially expressed between Col-0 and either pin6-2 or myb57-2.
Supporting Table 3. Analysis of pin6-2 nectar metabolites. Nectar was isolated via paper wick
from the lateral nectaries of Stage 14-15 flowers from 30-35 day old plants. Six biological
replicates were isolated from plants grown and analyzed as described in Methods. Metabolites
highlighted in green were significantly different between pin6-2 and Col-0.
Supporting Table 4. Analysis of gene expression associated with IAA synthetis and
inactivation/homeostasis. RNA-seq counts (from Supplemental Table 5) are presented for genes
known to be involved in IAA synthesis and inactivation/homeostasis. The genes listed for these
pathways were outlined in Mano and Nemoto, Journal of Experimental Botany, 2012, 63 (8):
2853-2872.
Supporting Table 5. Oligonucleotide primers used in this study.
Supporting Figure 1. PIN6 expression in mature flowers is not dependent on MYB57. RT
PCR analysis indicated PIN6 expression appears to be normal in myb57-2, and that MYB57
expression is unaltered in pin6-2 flowers. Expression of a strong nectary-specific gene required
for nectar production, SWEET9, is also unaltered in both myb57-2 and pin6-2 flowers. This
analysis was performed on RNA isolated from whole Stage 14-15 flowers, and was further
supported by RNAseq analysis of mature lateral nectaries (Table 2 and Supporting Table 3).
Supporting Figure 2. 3’ RACE analysis of PIN6 transcripts in pin6-1. A) Gel electrophoresis
of 3’ RACE products. B) Sequencing results of the 3’ RACE products (arrowhead) from panel
A. ^ indicates the Exon5/6 junction site. The pin6-1 T-DNA occurs in intron 5 (between exon
5/6). The predicted stop codon is underlined in red. C) Graphical representation of 3’ RACE
sequencing results aligned against the full-length PIN6 cDNA.
Supporting Figure 3. Graphic of RNAseq counts for PIN6 in pin6-2 and Col-0 from RNA
isolated from mature lateral nectaries. Full RNA count data is available in Supplemental
Table 2.
Supporting Figure 4. Complementation phenotypes of pin6-2 and myb57-2. A) pin6-2 was
complemented with PIN6pro:PIN6-GFP (see Figure 5); note the fully expanded petals (light
microscopy), restoration of short stamens (SS) and wild-type lateral nectaries (LN). B) myb57-2
was complemented with a full length genomic copy of the gene, which eliminated the petaloid
short stamen phenotype in all flowers (arrowhead). The lateral nectaries at the base of these short
stamen also appeared to be wild-type.
Supporting Figure 5. DR5:GUS in pin6 mutant backgrounds. Significantly reduced auxindependent DR5:GUS signal was observed in the lateral nectaries of pin6-1 and pin6-2, which is
consistent with what was observed in pin6 lines expressing DR5:GFP (Figure 6). Note: the
particular DR5:GUS/pin6-2 flower shown was missing median nectaries, which also
occasionally occurs in wild-type flowers.