Novel lncIPS1s and miR399s portend complex P-regulatory modules
in switchgrass
MicroRNAs (miRNAs) are small 21-22 nt short non-coding RNA molecules
that function in RNA silencing and posttranscriptional gene silencing.
MiRNAs are generated by cleavage of longer, poly-adenylated primarymiRNA transcripts that may be represented in normal RNA-seq
libraries. MiRNAs from the 399, 827 or 2111 families are crucial
components for P homeostasis (Hsieh et al., 2009, Pant et al., 2009),
and their primary transcripts were shown to be among the highly P-status
responsive transcripts in many plant species (reviewed in Scheible &
RojasāTriana, 2015; Lin et al., 2018).
Based on the number of sequencing reads, the identified primarymiR399 transcripts and IPS1 -like lncRNAs were among the
most highly expressed transcripts under mild, moderate and severe
P-stress in switchgrass, whereas a miR827 primary transcript was
only slightly induced. MiR2111 transcripts were completely absent
(Table S3; Figure 6 ), consistent with their absence in other
grasses (Scheible & Rojas-Triana, 2015). MiR399 is a well-characterized
regulator of shoot Pi accumulation under P-deprivation in all monocots
and dicots studied so far (Bari et al., 2006; Ajmera & Hodgman, 2019).
The identity of miR399s in the switchgrass genome was confirmed by
predicting the hairpin secondary structures of precursors, and comparing
the homology of premature and mature nucleotide sequences
(Dataset S1 ) with described miR399s. The number ofMiR399 genes identified in switchgrass is comparable to those
reported in other species, although the number (5 to 15 genes), and
their chromosomal arrangement can vary (Table S3 ) (Bari et al.,
2006; Hackenberg et al., 2013; Xu et al., 2013; Huen et al., 2018).
In P-stressed plants, target mimicry by IPS1 is an endogenous
mechanism used to confine the activity of miR399 through the production
of a decoy that is recognized and bound, but not cleaved
(Franco-Zorrilla et al., 2007). Switchgrass IPS1 -like
lncRNAs exhibit a highly conserved 23-nt-long motif with almost perfect
complementarity to miR399s, necessary for effective target mimicry
(Figure 6 ). This result suggests that miR399 activities are
subject to IPS1 -like lncRNAs inhibition as reported for
Arabidopsis (Franco-Zorrilla et al., 2007).
Among the 13 predicted miR399 target genes, two UBC24 /PHO2homologs were identified. This extends to perennial plants our
appreciation of miR399/PHO2 modules as central regulatory modules of P
homeostasis (Bari et al, 2006; Ouyang et al., 2016). A phosphate
transporter gene was also among the predicted miR399 targets. Phosphate
transporters as direct targets of miR399s have been verified or
computationally predicted also in other species (Devers et al., 2011; Xu
et al., 2013; Huen et al., 2018). These and our findings further
underline the importance of miR399s in regulation of plant Pi transport
and homeostasis. Aminocyclopropane-1-carboxylic acid synthase (ACS), a
rate-limiting enzyme in ethylene biosynthesis, is a surprising predicted
target of miR399, which suggests reduced ethylene synthesis during
P-limitation in switchgrass, in contrast to other plants (cf. Scheible
& Rojas-Triana, 2015). Interestingly, mutations in rice ACSgenes alter physiological and transcriptional responses associated with
P deficiency (Lee et al., 2019). MiR399-mediated downregulation of ACS
isoforms might fine-tune or prevent P-limitation responses in specific
switchgrass organs or tissues. Two switchgrass CAX genes encoding
putative vacuolar Ca2+/H+antiporters are also likely miR399 targets. Given the proposed
regulatory role of CAX proteins in suppressing expression of P genes
including phosphate transporters (Liu et al., 2011), it is conceivable
that calcium homeostasis/signaling also contributes to regulation of
P-stress responses in switchgrass.
Among the nine miR399 targets with detectable expression, seven did not
show a decrease, but several exhibited increases of transcript abundance
during P-limitation. While this behavior does not lend support to
miR399-mediated transcript cleavage per se , it also does not
preclude it. Targeted qRT-PCR assays that investigate transcript
cleavage at the miRNA-binding site or degradome analysis (Huen et al.,
2018) would be more informative in this regard. It is also possible that
translational repression of the target genes (Brodersen et al., 2008) is
an important mode of action for miR399, as previously suggested (Bari et
al., 2006). In summary, the number and variety of miR399s,IPS1 -like lncRNAs and putative target genes predicts a complex
pattern of post-transcriptional gene regulation in switchgrass under
P-stress.
In summary, this work represents the first integrated multi-level study
of switchgrass responses to P-limitation. The results reveal that
massive shifts in transcription underpin physiological and developmental
acclimation and adaptation to P-stress, and serve as a foundation for
future work aimed at improving P-acquisition and P-utilization
efficiency in this and other perennial grasses of economic value.