FIGURE LEGENDS
Figure 1. Effect of different P treatments on
switchgrass growth.
(a) Representative shoots of plants that received 600, 200, 60
or 20 µM Pi during growth for 28 days. Numbers in the photograph
indicate average height in centimeters ± standard error (n=5).(b) Shoot and root biomass in grams dry weight. (c)Inorganic phosphate concentration (mg per g fresh weight) for each
treatment. Data in (b, c) represent averages ± standard error
(n=5). Significance is indicated with different letters within same
plant organ (P ≤0.05).
Figure 2. Effect of P treatment on root growth and
morphology.
(a-d) Representative whole root morphology of plants treated
with: (a) 600; (b) 200; (c) 60; or(d) 20 µM Pi. Scale bars are 5 cm. (e) Primary root
length; (f) Total root length i.e. the sum of the lengths of
all roots; (g) Root surface area i.e. the sum of the surface
area of all roots. Data in (e-g) represent averages ± standard
error (n=5). Significance is indicated with different letters within
same plant organ (P ≤0.05). (h-k) Representative root
hair density/length phenotype of plants treated with: (h) 600;(i) 200; (j) 60; and (k) 20 µM phosphate.
Scale bars in (h) through (k) are 500 micrometer (µM).
Figure 3 . Effect of Pi concentration on ion and
metabolite abundances.
(a) Cation and anion concentrations (average ± standard error;
n=3) in plants treated with 600, 200, 60 or 20 µM Pi. (b)Effect of decreasing Pi supply on the abundance of major membrane lipids
and triacylglycerol. Relative abundances (average ± standard error;
n=3), as deduced from peak heights, are given for shoots (top panel) and
roots (bottom panel) of plants treated with 600 (blue bars), 200 (orange
bars), 60 (grey bars) or 20 µM (gold bars) phosphate. Major membrane
lipids shown are the phospholipids phosphatidylcholine (PC),
phosphatidylethanolamine (PE) and phosphatidylserine (PS), the
glycolipids monogalactosyl diacylglycerol (MGDG) and digalactosyl
diacylglycerol (DGDG), the chloroplast sulfolipid sulphoquinovosyl
diacylglycerol (SQDG), as well as the neutral storage lipid
triacylglycerol (TAG). (c) Principal component analysis (PCA)
of metabolite profiles based on relative abundance (average ± standard
error; n=3). All abundances were normalized to a range between 0 and 1
using Hellinger Transformation .
Figure 4 . Heat-map of metabolite profiles.
Metabolite changes (log2 scale) of 200, 60 or 20 µM Pi
supply are relative to 600 µM control in shoots and roots, respectively.
Shades of red or blue indicate increase or decrease in
Log2FC value, respectively.
Figure 5. Differentially expressed gene transcripts.
Depicted are the numbers of induced or repressed DEGs in plants that
received 200, 60 or 20 µM Pi in nutrient solutions, relative to control
plants (600 µM Pi). (a) DiVenn diagrams of transcripts in
shoots and roots with P adj value ≤0.05 and ≥ 3 fold-change (FC).(b) Summary of unique and common transcripts in shoots and
roots with P adj value ≤0.05 and ≥ 2, ≥ 3, ≥ 5 and ≥ 10 FC.(c) Gene Ontology enrichment analysis of DEGs with P adj
value ≤0.05 and ≥ 2-fold change. Red and blue colors indicate induced
and repressed genes, respectively. Yellow nodes in A indicate inverse
responses, i.e. rare induction in one sample but repression in the
other.
Figure 6 . Identified miR399s, IPS1-likesequences and putative target gene transcripts.
(a) Alignment, phylogenetic relationship and consensus sequence
(sequence logo) of miR399 sequences found in 15 unannotated switchgrass
DEG transcripts. Refer to Table S1 for the strong responses of the
primary miR399 transcripts to Pi limitation. Depicted below the
alignment are segments of putative IPS1-like, long non-coding RNAs
(lncRNAs) and their base pairing with the miR399 consensus sequence.
Blue colored nucleotides are the central mismatched loops critical for
resistance to miR399-guided cleavage, thus enabling miR399
sequestration. (b) Responses of identified IPS1-liketranscripts and predicted miR399 target gene transcripts to P stress
(200, 60 or 20 µM Pi relative to the 600 µM Pi control) in shoot and
root as deduced from RNA-Seq.