Global transcriptional changes in response to increasing P-limitation
Analysis of RNA Seq data identified a total of 99,409 different transcripts, called MSTRG.#####, 68,573 of which mapped to reference genes, denoted Pavir.#KG######. These transcripts were further analyzed to identify differentially-expressed gene transcripts (DEGs) in six comparisons (i.e. shoots or roots, at the three P-stress levels (200, 60 and 20 µM Pi) relative to control (600 µM Pi), following filtering by three criteria: (1) expression strength, eliminating those with an average FPKM value less than 3 in at least one of the two conditions compared; (2) minimum fold change (FC), of. 2-, 3-, 5- or 10-fold; and (3) P -value (P adj≤0.05) calculated from the FPKM values from three replicates. The first criterion reduced the number of transcripts considered by 70-75% for each of the six comparisons, while the inclusion of minimum fold-change and the P-value criteria reduced the numbers of DEGs to those shown in Table S2and Figure S1 . The number of DEGs increased with the degree of P-stress, with several thousand genes being either induced or repressed ≥3-fold relative to P-replete controls under moderate and severe P-stress (Figure 5a ). Most of DEGs identified in mild- and moderately-stressed plants were also found in moderately or severely P-stressed plants, respectively, indicating that mechanisms of acclimation to stress were sustained and expanded with increasing stress (Figure 5b; Table S2 ). DiVenn analysis of DEGs indicated that shoots and roots shared some transcriptional responses to P-stress, although many more were unique to either shoots or roots (Figure S2 ).
To gain a general understanding of the biological processes affected by P-stress, gene ontology (GO) enrichment analyses of DEGs was conducted. Among the processes induced by P-stress, transmembrane transport of anions, including phosphate and nitrogen compounds, as well as lipid metabolism/catabolism and glycolipid synthesis stood out in shoots and roots. Other processes appeared to be more organ-specific. In roots, lignin, phenylpropanoid, and terpenoid catabolism/metabolism were amongst the induced GO categories, while in shoots response to nutrient levels and starvation, chloroplast RNA modification, nitrate transport, and glyoxylate cycle were amongst the most pronounced processes affected by P-stress. As for repressed processes, regulation of transcription and responses to stimuli, hormones and stresses were over-represented in shoots and roots, while photosynthesis and redox homeostasis were specific to shoots (Figure 5c ).