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 ).