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.