MicroRNAs (miRNAs) are small, endogenous RNAs that regulate gene expression in plants and animals. In plants, these approximately 21-nucleotide RNAs are processed from stem-loop regions of long primary transcripts by a Dicer-like enzyme and are loaded into silencing complexes, where they generally direct cleavage of complementary mRNAs. Although plant miRNAs have some conserved functions extending beyond development, the importance of miRNA-directed gene regulation during plant development is now particularly clear. Identified in plants less than four years ago, miRNAs are already known to play numerous crucial roles at each major stage of development-typically at the cores of gene regulatory networks, targeting genes that are themselves regulators, such as those encoding transcription factors and F-box proteins.

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MicroRNAs AND THEIR REGULATORY ROLES IN PLANTS

https://www.cell.com/cell/pdf/S0092-8674(09)00128-7.pdf

Origin, biogenesis, and activity of plant microRNAs.

MicroRNAs (miRNA) regulate key aspects of development and physiology in animals and plants. These regulatory RNAs act as guides of effector complexes to recognize specific mRNA sequences based on sequence complementarity, resulting in translational repression or site-specific cleavage. In plants, most miRNA targets are cleaved and show almost perfect complementarity with the miRNAs around the cleavage site. Here, we examined the non-protein coding gene IPS1 (INDUCED BY PHOSPHATE STARVATION 1) from Arabidopsis thaliana. IPS1 contains a motif with sequence complementarity to the phosphate (Pi) starvation-induced miRNA miR-399, but the pairing is interrupted by a mismatched loop at the expected miRNA cleavage site. We show that IPS1 RNA is not cleaved but instead sequesters miR-399. Thus, IPS1 overexpression results in increased accumulation of the miR-399 target PHO2 mRNA and, concomitantly, in reduced shoot Pi content. Engineering of IPS1 to be cleavable abolishes its inhibitory activity on miR-399. We coin the term 'target mimicry' to define this mechanism of inhibition of miRNA activity. Target mimicry can be generalized beyond the control of Pi homeostasis, as demonstrated using artificial target mimics.

/pdf/target-mimicry-provides-a-new-mechanism-for-regulation-of-exf8bgsdqb.pdf

Target mimicry provides a new mechanism for regulation of microRNA activity

Plant responses to different stresses are highly complex and involve changes at the transcriptome, cellular, and physiological levels. Recent evidence shows that plants respond to multiple stresses differently from how they do to individual stresses, activating a specific programme of gene expression relating to the exact environmental conditions encountered. Rather than being additive, the presence of an abiotic stress can have the effect of reducing or enhancing susceptibility to a biotic pest or pathogen, and vice versa. This interaction between biotic and abiotic stresses is orchestrated by hormone signalling pathways that may induce or antagonize one another, in particular that of abscisic acid. Specificity in multiple stress responses is further controlled by a range of molecular mechanisms that act together in a complex regulatory network. Transcription factors, kinase cascades, and reactive oxygen species are key components of this cross-talk, as are heat shock factors and small RNAs. This review aims to characterize the interaction between biotic and abiotic stress responses at a molecular level, focusing on regulatory mechanisms important to both pathways. Identifying master regulators that connect both biotic and abiotic stress response pathways is fundamental in providing opportunities for developing broad-spectrum stress-tolerant crop plants.

The interaction of plant biotic and abiotic stresses: from genes to the field

To better understand the diversity of small silencing RNAs expressed in plants, we employed high-throughput pyrosequencing to obtain 887,000 reads corresponding to Arabidopsis thaliana small RNAs. They represented 340,000 unique sequences, a substantially greater diversity than previously obtained in any species. Most of the small RNAs had the properties of heterochromatic small interfering RNAs (siRNAs) associated with DNA silencing in that they were preferentially 24 nucleotides long and mapped to intergenic regions. Their density was greatest in the proximal and distal pericentromeric regions, with only a slightly preferential propensity to match repetitive elements. Also present were 38 newly identified microRNAs (miRNAs) and dozens of other plausible candidates. One miRNA mapped within an intron of DICER-LIKE 1 (DCL1), suggesting a second homeostatic autoregulatory mechanism for DCL1 expression; another defined the phase for siRNAs deriving from a newly identified trans-acting siRNA gene (TAS4); and two depended on DCL4 rather than DCL1 for their accumulation, indicating a second pathway for miRNA biogenesis in plants. More generally, our results revealed the existence of a layer of miRNA-based control beyond that found previously that is evolutionarily much more fluid, employing many newly emergent and diverse miRNAs, each expressed in specialized tissues or at low levels under standard growth conditions.

/pdf/a-diverse-and-evolutionarily-fluid-set-of-micrornas-in-4uxgt9gjuk.pdf

A diverse and evolutionarily fluid set of microRNAs in Arabidopsis thaliana

https://www.cell.com/current-biology/pdf/S0960-9822(05)01216-9.pdf

A miRNA involved in phosphate-starvation response in Arabidopsis

In this study, we reveal a mechanism by which plants regulate inorganic phosphate (Pi) homeostasis to adapt to environmental changes in Pi availability. This mechanism involves the suppression of a ubiquitin-conjugating E2 enzyme by a specific microRNA, miR399. Upon Pi starvation, the miR399 is upregulated and its target gene, a ubiquitin-conjugating E2 enzyme, is downregulated in Arabidopsis thaliana. Accumulation of the E2 transcript is suppressed in transgenic Arabidopsis overexpressing miR399. Transgenic plants accumulated five to six times the normal Pi level in shoots and displayed Pi toxicity symptoms that were phenocopied by a loss-of-function E2 mutant. Pi toxicity was caused by increased Pi uptake and by translocation of Pi from roots to shoots and retention of Pi in the shoots. Moreover, unlike wild-type plants, in which Pi in old leaves was readily retranslocated to other developing young tissues, remobilization of Pi in miR399-overexpressing plants was impaired. These results provide evidence that miRNA controls Pi homeostasis by regulating the expression of a component of the proteolysis machinery in plants.

Regulation of Phosphate Homeostasis by MicroRNA in Arabidopsis

Recent studies have demonstrated the important role of plant microRNAs (miRNAs) under nutrient deficiencies. In this study, deep sequencing of Arabidopsis (Arabidopsis thaliana) small RNAs was conducted to reveal miRNAs and other small RNAs that were differentially expressed in response to phosphate (Pi) deficiency. About 3.5 million sequence reads corresponding to 0.6 to 1.2 million unique sequence tags from each Pi-sufficient or Pi-deficient root or shoot sample were mapped to the Arabidopsis genome. We showed that upon Pi deprivation, the expression of miR156, miR399, miR778, miR827, and miR2111 was induced, whereas the expression of miR169, miR395, and miR398 was repressed. We found cross talk coordinated by these miRNAs under different nutrient deficiencies. In addition to miRNAs, we identified one Pi starvation-induced DICER-LIKE1-dependent small RNA derived from the long terminal repeat of a retrotransposon and a group of 19-nucleotide small RNAs corresponding to the 5' end of tRNA and expressed at a high level in Pi-starved roots. Importantly, we observed an increased abundance of TAS4-derived trans-acting small interfering RNAs (ta-siRNAs) in Pi-deficient shoots and uncovered an autoregulatory mechanism of PAP1/MYB75 via miR828 and TAS4-siR81(-) that regulates the biosynthesis of anthocyanin. This finding sheds light on the regulatory network between miRNA/ta-siRNA and its target gene. Of note, a substantial amount of miR399* accumulated under Pi deficiency. Like miR399, miR399* can move across the graft junction, implying a potential biological role for miR399*. This study represents a comprehensive expression profiling of Pi-responsive small RNAs and advances our understanding of the regulation of Pi homeostasis mediated by small RNAs.

Uncovering Small RNA-Mediated Responses to Phosphate Deficiency in Arabidopsis by Deep Sequencing

Plants acquire phosphorus in the form of phosphate (Pi), the concentration of which is often limited for plant uptake. Plants have developed diverse responses to conserve and remobilize internal Pi and to enhance Pi acquisition to secure them against Pi deficiency. These responses are achieved by the coordination of an elaborate signaling network comprising local and systemic machineries. Recent advances have revealed several important components involved in this network. Pi functions as a signal to report its own availability. miR399 and sugars act as systemic signals to regulate responses occurring in roots. Hormones also play crucial roles in modulating gene expression and in altering root system architecture. Transcription factors function as a hub to perceive the signals and to elicit steady outputs. In this review, we outline the current knowledge on this subject and present hypotheses pertaining to other potential signals and to the organization and coordination of signaling.

Signaling Network in Sensing Phosphate Availability in Plants

We recently demonstrated that microRNA399 (miR399) controls inorganic phosphate (Pi) homeostasis by regulating the expression of UBC24 encoding a ubiquitin-conjugating E2 enzyme in Arabidopsis (Arabidopsis thaliana). Transgenic plants overexpressing miR399 accumulated excessive Pi in the shoots and displayed Pi toxic symptoms. In this study, we revealed that a previously identified Pi overaccumulator, pho2, is caused by a single nucleotide mutation resulting in early termination within the UBC24 gene. The level of full-length UBC24 mRNA was reduced and no UBC24 protein was detected in the pho2 mutant, whereas up-regulation of miR399 by Pi deficiency was not affected. Several characteristics of Pi toxicity in the pho2 mutant were similar to those in the miR399-overexpressing and UBC24 T-DNA knockout plants: both Pi uptake and translocation of Pi from roots to shoots increased and Pi remobilization within leaves was impaired. These phenotypes of the pho2 mutation could be rescued by introduction of a wild-type copy of UBC24. Kinetic analyses revealed that greater Pi uptake in the pho2 and miR399-overexpressing plants is due to increased Vmax. The transcript level of most PHT1 Pi transporter genes was not significantly altered, except PHT1;8 whose expression was enhanced in Pi-sufficient roots of pho2 and miR399-overexpressing compared with wild-type plants. In addition, changes in the expression of several organelle-specific Pi transporters were noticed, which may be associated with the redistribution of intracellular Pi under excess Pi. Furthermore, miR399 and UBC24 were colocalized in the vascular cylinder. This observation not only provides important insight into the interaction between miR399 and UBC24 mRNA, but also supports their systemic function in Pi translocation and remobilization.

Shu-I Lin

Papers

A miRNA involved in phosphate-starvation response in Arabidopsis

Regulation of Phosphate Homeostasis by MicroRNA in Arabidopsis

Uncovering Small RNA-Mediated Responses to Phosphate Deficiency in Arabidopsis by Deep Sequencing

Signaling Network in Sensing Phosphate Availability in Plants

pho2, a phosphate overaccumulator, is caused by a nonsense mutation in a microRNA399 target gene.