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 & Rojas‐Triana, 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.