Switchgrass miR399s, their putative target genes, and IPS1-like lncRNAs
Although miRNAs from the 399, 827 or 2111 families were previously found to be induced by P-limitation and have been implicated in the regulation of P-stress responses in multiple plant species, none were found among the annotated switchgrass DEGs. Therefore, we searched specifically for DEGs containing sequence motifs / signatures of these miRNAs, before confirming that matching sequences were part of predicted stable RNA stem-loop structures. A total of 15 un-annotated transcripts containing miR399 sequences with predicted stable stem-loop structures (data not shown) were found (Dataset S1; cf. miRBase at www.mirbase.org). Many of these transcripts were strongly induced under mild, moderate and severe P-stress (Table S3 ). For example, in the set of 380 strongly-induced (> 5-fold) DEGs found in shoots of plants treated with 60 µM P, ten primary transcripts for miR399s were found (Table S4 ). One potential primary transcript for miR827 was also found (Table S3 ), but none for miR2111. Phylogenetic analysis and alignment of the fifteen 21-nt long miR399 sequences grouped them into several subfamilies, named miR399-1 to miR399-6, that are predominantly defined by polymorphisms in positions 13 and 14 (Figure 6a ).
Target mimicry by IPS1 , a long, non-coding RNA (lncRNA), is a mechanism for controlling miR399 activity in Arabidopsis (Franco-Zorrilla et al., 2007). We found three unannotated switchgrassIPS1 -like lncRNA transcripts (ISP1 -like1,ISP1 -like2a and ISP1 -like2b) with base complementarity to miR399s and the presence of a critical, central 3-nt mismatched loop (cf. Figure 6a ) that allows binding to miR399 but prevents miR399-guided lncRNA cleavage in the Dicer complex. Like AtIPS1in Arabidopsis, the three switchgrass IPS1 -like transcripts were also highly induced under mild, moderate and severe P-stress in shoot and root (Figure 6b ).
Using psRNATarget (Dai et al., 2018), we identified 13 potential miR399 target transcripts with sequence complementarity to miR399s (Figure 6b ). Members of subfamily miR399-3 and miR399-4 had almost perfect complementarity to five or six potential binding sites in the 5’-UTR regions of the two UBC24 /PHO2 homologs identified (Bari et al., 2006; Figure S3 ). Three other DEG transcripts that are putative targets of the miR399-3 subfamily genes encode an inorganic P-transporter, an aminocyclopropane-1-carboxylase synthase ACS9/ETO3 homolog, and an unknown protein. Three DEG transcripts encoding a peptidase and two proteins of unknown function are targets for the miR399-2 subfamily. Finally, five DEG transcripts encoding two Ca2+/H+ (CAX) antiporters, a UDP-Rha/UDP-Gal transporter, and a non-specific serine/threonine-protein kinase are the most likely targets of the miR399-6 subfamily (Figure S3 ).