Transcriptional hallmark responses to P limitation conserved in switchgrass
Previous research with other plant species identified conserved transcriptional responses to P-stress (e.g. Misson et al., 2005; Bari et al., 2006; Morcuende et al., 2007; Pant et al., 2009; Plaxton & Tran, 2011; Secco et al., 2013). Switchgrass exhibited many of the same transcriptional responses to P limitation (Table S3 ). These responses included induction of gene transcripts for (i) uptake and transport of Pi and other inorganic ions,e.g. Pht1 , PHO1 ,KUP , NRT1 ; (ii) Pi salvage systems,e.g. PAPs , ribonucleases, GDPDs , phospholipid degradation genes; (iii) alternative metabolic pathways that lower P requirements, e.g. inorganic pyrophosphatases); (iv) sulfo- and glycolipid synthesis, e.g.MGD s, DGD s, SQD ; (v) redirection of carbon metabolism; (vi) phytohormone synthesis/response pathways; (vii) disease/pathogen stress–responses, e.g. Cysteine-rich RLK, CAPs; and(viii) repression of gene transcripts associated with the photosynthetic machinery and photosynthesis (TableS3 ).
Surprisingly, few DEGs encoding SPX-domain proteins and no microRNA399s were among the annotated switchgrass transcripts, possibly reflecting incomplete annotation of switchgrass genes. In fact, unannotated DEGs represented between one-third and two-thirds of all P-responsive gene transcripts, depending on treatment and cut-off values used (Figure S1 ). Therefore, we investigated the many unannotated DEGs through manual homology searches against transcripts/proteins from other plant species. To this end, we chose strongly responsive (>5-fold change) DEGs in shoots and roots of moderately-stressed plants grown with 60 µM Pi supply, which likely elicited many specific P-stress responses without triggering many non-specific responses related to severe impairment of growth and development (cf. Figure 5b; Figure S1; Table S4 ).
Among the 380 DEGs with at least 5-fold change in transcript abundance in shoots, 234 were up-regulated and 146 down-regulated. Of the up-regulated DEGs, 109 (47%) were annotated, and many of these were homologous to P-responsive DEGs in other plant species (B. distachyon , M. truncatula , O. sativa and A. thaliana ). Some of these genes have established functions during P-limitation, such as phosphate transporters or purple acid phosphatases (Table S4 ), but many encode unknown or uncharacterized proteins, or proteins with functions not previously linked to P-limitation, such as three DEGs encoding Sua5/YciO/YrdC/YwlC proteins required for tRNA modification (Yacoubi et al., 2009). Moreover, 127 up- or down-regulated DEGs with gene identifier, showed no homology to proteins or RNAs in other species. These observations point to unknown, possibly switchgrass-specific transcriptional P-starvation responses. For the 125 up-regulated, unannotated DEGs, we performed dedicated, manual BLAST searches against genes from other species (clade Viridiplantae). This revealed that 84 of these DEGs do have significant homology to known proteins, many of which are encoded by P- responsive genes. This includes six additional SPX-domain proteins, four glycerophosphodiester phosphodiesterases, three purple acid phosphatases, two PHO1-like phosphate transporters, two monogalactosyldiacylglycerol synthases (MGD , involved in glycolipid synthesis), two sulfoquinovosyl transferases (SQD , sulfolipid synthesis), another phosphoenolpyruvate carboxykinase, two glucose-1-phosphate adenylyltransferases small subunits and an ADP-glucose pyrophosphorylase required for starch synthesis (Table S4 ). For 31 (25%) of the 125 up-regulated, unannotated DEGs no protein homologs were found. It is possible that some of these 31 DEGs represent P-status responsive microRNA primary transcripts or other long non-coding RNAs (e.g. IPS1, TAS4;Franco-Zorrilla et al., 2007; Hsieh et al., 2009) that play important roles during P-limitation, but can only be found with targeted searches (see below), while other DEGs may represent novel aspects of P-starvation responses in plants.
We also scrutinized strongly-responsive DEGs (> 5-fold change) in roots of plants grown with 60 µM Pi. Among the up-regulated annotated DEGs in roots were many related to well-known transcriptional responses to P-stress, e.g. membrane lipid remodeling, phosphate uptake and salvaging. An interesting observation was the upregulation of three ent-copalyl diphosphate synthases, an ent-kaurenoic acid oxidase-like protein and an ent-kaurenoic acid hydroxylase, all involved in gibberellin biosynthesis (Table S4 ).