Phosphate Deficiency: A Tale from the End of PILNCR2

A deficiency in inorganic phosphate (Pi) induces the expression of miRNA399 and the accumulation of its target Pi transporters (PHT1s) mRNA, which is contrary to the goal of miRNA-mediated gene regulation. Recently, a novel mechanism of RNA/RNA-duplex formation between the transcripts of a Pi deficiency-induced long non-coding RNA (PILNCR2) and PHT1s has been reported, which prevents the binding and cleavage of miRNA399 to PHT1 mRNAs, thereby providing tolerance of Pi-deficient conditions. Moreover, the way in which ribosomes move through the RNA/RNA-duplex for the translation of PHT1 transporter proteins remains elusive.


Introduction
Plants obtain phosphorus (P) as inorganic phosphate (Pi) from the soil to enable their growth. However, the largest proportion of agricultural areas shows Pi deficiency due to its slow diffusion, which restrains crop productivity [1]. Various Pi transporters (PHTs) and transcription factors, like Phosphate Starvation Response (PHRs) and Sensitive to Proton Rhizotoxicity 1 (STOP1), are involved in the transportation and regulation of Pi in plants [2][3][4]. An ubiquitin-conjugating E2 enzyme (PHO2) inhibits PHTs and phosphate 1 (PHO1) transporters to reduce Pi uptake. In contrast, miR399, after inducing expression during conditions of Pi deficiency, suppresses PHO2 expression during Pi homeostasis [5]. In addition, Du et al. [5] showed that Pi deficiency-induced lncRNA (PILNCR1) could protect ZmPHO2 mRNAs by inhibiting ZmmiRNA399. Moreover, miRNA399 has also been assumed to target PHT genes in maize [6], which suggests that additional investigation is required to establish the mechanism of Pi homeostasis, particularly in maize. Recently, Wang et al. [7] revealed the lncRNA (PILNCR2)-mediated regulation of interactions between miRNA399 and PHT1 genes during Pi homeostasis in maize.

miRNA399 Targets PHT1 Family Genes
The PHT1 is a multigene family in maize that comprises 13 members, of which five genes (PHT1;1, 1;3, 1;7, 1;8, and 1;13) have been predicted as potential miRNA399 targets [6,8]. Since Pi deficiency induces ZmmiRNA399 expression, it is believed that the target genes should be downregulated. Therefore, Wang et al. [7] first analysed the expression of target ZmPHT1 genes in the root tissues using strand-specific RNA libraries generated in Pi-deficient conditions from two inbred lines (CCM454 and 31778) [9] and further validated via quantitative real-time PCR (qRT-PCR). Contrary to our expectations, the results revealed upregulated expression of three ZmPHT1 genes (ZmPHT1;1, 1;3, and 1;13) under conditions of Pi deficiency, which raised concerns about the validity of targeting ZmPHT1 genes by ZmmiRNA399. Therefore, this study then validated the interaction between ZmmiRNA399 and mRNAs of ZmPHT1 genes. The transient co-expression assays revealed reduced accumulation of ZmPHT1 transcripts in the presence of miRNA399b in Nicotiana benthamiana. In addition, the reduced expression of ZmPHT1;1, 1;3, and 1;13 genes was reported in the transgenic maize that overexpress the MIRNA399b [5]. Further, these ZmPHT1 genes comprised potential cleavage sites for miRNA399. These results assured that the mRNAs of ZmPHT1 genes are potential targets for miRNA399. Moreover, the Pi deficiency-induced upregulated expression of both miRNA399 and its target ZmPHT1 genes indicates the existence of an alternate regulatory pathway/mechanism.

PILNCR2 Regulates miRNA399 Interaction with ZmPHT1 Genes
In an earlier study, PILNCR1-mediated regulation of Pi homeostasis was reported, in which PILNCR1 was shown to protect ZmPHO2 mRNAs by inhibiting their cleavage by ZmmiRNA399 [5]. Therefore, Wang et al. [7] explored the lncRNA libraries of CCM454 and 31,778 inbred lines of maize generated under conditions of Pi deficiency [5], and identified PILNCR2 as a cis-natural antisense transcript (NAT) that has a complementary sequence to ZmPHA1;1. Analysis of full-length cDNA sequences obtained via RACE and calf intestine alkaline phosphatase (CIP) treatment suggested the occurrence of 5 -capping and 3 -poly-A tails in PILNCR2, indicating its RNA polymerase II-mediated transcription. Further, none of the ORFs predicted in the PILNCR2 sequence could encode functional protein, which confirmed that it is a lncRNA. The expression profiling suggested the upregulation of PIL-NCR2 under Pi deficiency, and northern blotting confirmed its localisation in the cytoplasm. The in situ hybridisation indicated the co-occurrence of PILNCR2 and ZmPHT1;1, 1;3, and 1;13 transcripts throughout the root. These findings suggested the coregulation of PILNCR2 and ZmPHT1s via an elusive mechanism.
The cis-NATs usually form dsRNA or siRNAs from the overlapping region (OR) of a gene pair to regulate gene expression [10]. Moreover, the current study could not recognize siRNA that originated from PILNCR2 or ZmPHT1;1 ORs. The RNA-RNA binding assays performed in in vitro conditions using BrU-labelled PILNCR2 RNA and anti-BrdU mAb antibodies revealed higher accumulations of ZmPHT1;1, 1;3, and 1;13 transcripts when incubated with overlapping PILNCR2, thereby suggesting the formation of a RNA/RNA duplex between PILNCR2 and ZmPHT1 transcripts. The in vivo ribonuclease protection assay confirmed the above interaction. Additionally, the co-expression analyses of ZmMIR399b combined with ZmPHT1;1, 1;3, or 1;13 in the presence and absence of PILNCR2 were performed for N. benthamiana. The qRT-PCR analyses showed that higher accumulations of mRNAs of ZmPHT1;1, 1;3, or 1;13 were present for PILNCR2, suggesting that they were protected from ZmMIR399b-mediated cleavage. Even the mutations or deletions in ZmmiR399-overlapping regions in PILNCR2 were not able to inhibit duplex formation. The RNA/RNA duplex, which was generated between the transcripts of PILNCR2 and ZmPHT1s, interferes with the binding of ZmmiRNA399 to the mRNAs of the ZmPHT1 family of genes, ultimately protecting them from miRNA-mediated cleavage ( Figure 1). assured that the mRNAs of ZmPHT1 genes are potential targets for miRNA399. Moreover, the Pi deficiency-induced upregulated expression of both miRNA399 and its target ZmPHT1 genes indicates the existence of an alternate regulatory pathway/mechanism.

PILNCR2 Regulates miRNA399 Interaction with ZmPHT1 Genes
In an earlier study, PILNCR1-mediated regulation of Pi homeostasis was reported, in which PILNCR1 was shown to protect ZmPHO2 mRNAs by inhibiting their cleavage by ZmmiRNA399 [5]. Therefore, Wang et al. [7] explored the lncRNA libraries of CCM454 and 31,778 inbred lines of maize generated under conditions of Pi deficiency [5], and identified PILNCR2 as a cis-natural antisense transcript (NAT) that has a complementary sequence to ZmPHA1;1. Analysis of full-length cDNA sequences obtained via RACE and calf intestine alkaline phosphatase (CIP) treatment suggested the occurrence of 5'-capping and 3'-poly-A tails in PILNCR2, indicating its RNA polymerase II-mediated transcription. Further, none of the ORFs predicted in the PILNCR2 sequence could encode functional protein, which confirmed that it is a lncRNA. The expression profiling suggested the upregulation of PILNCR2 under Pi deficiency, and northern blotting confirmed its localisation in the cytoplasm. The in situ hybridisation indicated the co-occurrence of PILNCR2 and ZmPHT1;1, 1;3, and 1;13 transcripts throughout the root. These findings suggested the coregulation of PILNCR2 and ZmPHT1s via an elusive mechanism.
The cis-NATs usually form dsRNA or siRNAs from the overlapping region (OR) of a gene pair to regulate gene expression [10]. Moreover, the current study could not recognize siRNA that originated from PILNCR2 or ZmPHT1;1 ORs. The RNA-RNA binding assays performed in in vitro conditions using BrU-labelled PILNCR2 RNA and anti-BrdU mAb antibodies revealed higher accumulations of ZmPHT1;1, 1;3, and 1;13 transcripts when incubated with overlapping PILNCR2, thereby suggesting the formation of a RNA/RNA duplex between PILNCR2 and ZmPHT1 transcripts. The in vivo ribonuclease protection assay confirmed the above interaction. Additionally, the co-expression analyses of ZmMIR399b combined with ZmPHT1;1, 1;3, or 1;13 in the presence and absence of PILNCR2 were performed for N. benthamiana. The qRT-PCR analyses showed that higher accumulations of mRNAs of ZmPHT1;1, 1;3, or 1;13 were present for PILNCR2, suggesting that they were protected from ZmMIR399b-mediated cleavage. Even the mutations or deletions in ZmmiR399-overlapping regions in PILNCR2 were not able to inhibit duplex formation. The RNA/RNA duplex, which was generated between the transcripts of PILNCR2 and ZmPHT1s, interferes with the binding of ZmmiRNA399 to the mRNAs of the ZmPHT1 family of genes, ultimately protecting them from miRNA-mediated cleavage (Figure 1).  model that demonstrates the normal expression of PHT1 genes and PLNCR2 during conditions of Pi sufficiency, while Pi deficiency induces the expression of PHT1 genes and ZmmiRNA399, along with the PLNCR2 from the opposite strand of PHT1;1. All transcripts translocate to the cytoplasm, where the PLNCR2 transcripts form RNA/RNA duplexes with the mRNAs of PHT1s due to their complementary sequences. The RNA duplex prevents the binding of ZmmiRNA399 to mRNAs of PHT1s, protecting them from cleavage. These results highlight the increased accumulation of PHT1 transcripts that form more PHT1s used for the transportation of Pi into the cytoplasm, thereby proving low Pi tolerance. The figure is adapted and modified from the study of Wang et al. [7], with permission given by Elsevier through Rightslink, licence number 5580761357750.

PILNCR2 Expression Promotes Low-Pi Tolerance
Since PILNCR2 protects ZmPHT1s from miRNA-mediated degradation and showed inducible expression under Pi-deficient conditions, one questions remain to be answered: is it involved in low-Pi tolerance? To answer this question, Wang et al. [7] generated overexpression (OE), knock-down (KD), and knock-out (KO) lines for PILNCR2 in the maize plants and analysed them for low Pi tolerance. The PILNCR2 OE provided significant tolerance in the maize plants under low-Pi conditions without accumulation of anthocyanin. However, both KO and KD lines showed higher accumulation of anthocyanin under low-Pi treatment than the wild type and OE lines. The Pi-depletion assay revealed a higher rate of Pi absorption by OE lines and increased accumulation of Pi in various plant parts. In contrast, KO and KD lines exhibited highly reduced Pi absorption rates and depleted P accumulation in roots and shoots, even in the Pi-sufficient condition. Moreover, PILNCR2 KO abolished Pi-toxicity caused by ZmmiRNA399b OE in the double transgenic maize under Pi-sufficient conditions. Du et al. [5] earlier reported that the OE of ZmMIRNA399b caused Pi toxicity by downregulating the expression of ZmPHO2. Additionally, Wang et al. [7] found that the expression level of ZmMIRNA399 could not be affected by both OE and KO of PILNCR2, though ZmPHT1 genes showed increased expression in OE and decreased expression in KO lines of PILNCR2 transgenic maize. Further, the ZmMIRNA399 OE did not affect PILNCR2 expression, though it reduced the accumulation of transcripts of ZmPHT1 genes. These results suggest the function of PILNCR2 in conditions of low Pi tolerance as being to protect the mRNAs of ZmPHT1 genes from ZmmiRNA399-mediated degradation.

Concluding Remarks and Future Perspective
In summary, PILNCR2 regulates Pi homeostasis in maize, and its induced expression provides low Pi tolerance by protecting the ZmmiRNA399-mediated cleavage of PHT1 mRNAs. The Pi deficiency-induced upregulation of both ZmmiRNA399 and its target PHT1 genes suggested an alternative mechanism of regulation. The PILNCR2 transcribed from the opposite strand of ZmPHT1;1 forms RNA/RNA duplex in combination with the mRNAs of ZmPHT1 genes, preventing binding of ZmmiRNA399. The study provides a novel mechanism for lncRNA-mediated regulation of gene expression. Moreover, the study raises a few interesting questions. Since the transcript of PILNCR2 completely masked the mRNAs of ZmPHT1s from the 5 end to the 3 end, how did ribosomes bind and move through the RNA/RNA duplex during translation? The mechanism of ribosome movement through RNA/RNA duplex would reveal a novel avenue for the functioning of lncRNAs, especially the NATs. Further, the accumulation of ZmPHT1 proteins could be analysed in tandem with their transcript abundance. This observation raises another question: is this method the only way to determine low Pi tolerance in plants? Alternatively, there may be some other transporters or regulatory mechanism available that function in coordination with this pathway; this possibility needs to be addressed in future studies.