A Role for Arabidopsis miR399f in Salt,Drought, and ABA Signaling

MiR399f plays a crucial role in maintaining phosphate homeostasis in Arabidopsis thaliana. Under phosphate starvation conditions, AtMYB2, which plays a role in plant salt and drought stress responses, directly regulates the expression of miR399f. In this study, we found that miR399f also participates in plant responses to abscisic acid (ABA), and to abiotic stresses including salt and drought. Salt and ABA treatment induced the expression of miR399f, as confirmed by histochemical analysis of promoter-GUS fusions. Transgenic Arabidopsis plants overexpressing miR399f (miR399f-OE) exhibited enhanced tolerance to salt stress and exogenous ABA, but hypersensitivity to drought. Our in silico analysis identified ABF3 and CSP41b as putative target genes of miR399f, and expression analysis revealed that mRNA levels of ABF3 and CSP41b decreased remarkably in miR399f-OE plants under salt stress and in response to treatment with ABA. Moreover, we showed that activation of stress-responsive gene expression in response to salt stress and ABA treatment was impaired in miR399f-OE plants. Thus, these results suggested that in addition to phosphate starvation signaling, miR399f might also modulates plant responses to salt, ABA, and drought, by regulating the expression of newly discovered target genes such as ABF3 and CSP41b.     

Long-distance movement and differential targeting of microRNA399s

We have previously demonstrated that miR399s control phosphate (Pi) homeostasis by regulating the expression of a ubiquitin-conjugating E2 enzyme (UBC24/PHO2) in Arabidopsis. Changes in miR399-dependent PHO2 gene expression modulate Pi uptake, allocation and remobilization. More recently, we provided evidence that miR399s are able to move in the phloem stream and across grafting junctions from the scions overexpressing miR399 to the wild-type rootstocks. Movement of miR399s serves as a long-distance signal to report and balance the Pi status between shoots and roots. Of note, results from grafting experiments indicate that miR399b is less efficient in cleaving the PHO2 mRNA than is miR399f, despite the similar mobility of the two miR399s. We propose that nucleotide 13 of miR399s, which gives rise to the sequence variation among different miR399 species, could be involved in regulating the abundance of PHO2 mRNA through sequence complementarity to the target sequences of PHO2 mRNA and mimicking target sequence of At4/IPS1 noncoding RNAs.Key words: phosphate, microRNA399, PHO2, UBC24, long-distance movement, At4/IPS1     

The role of the miR399-<Emphasis Type="Italic">PHO2</Emphasis> module in the regulation of flowering time in response to different ambient temperatures in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

A moderate change in ambient temperature significantly affects plant physiology including flowering time. MiR399 and its target gene PHOSPHATE 2 (PHO2) are known to play a role in the maintenance of phosphate homeostasis. However, the regulation of flowering time by the miR399-PHO2 module has not been investigated. As we have previously identified miR399 as an ambient temperature-responsive miRNA, we further investigated whether a change in expression of the miR399-PHO2 module affects flowering time in response to ambient temperature changes. Here, we showed that miR399b-overexpressing plants and a loss-of-function allele of PHO2 (pho2) exhibited an early flowering phenotype only at normal temperature (23°C). Interestingly, their flowering time at lower temperature (16°C) was similar to that of wild-type plants, suggesting that alteration in flowering time by miR399 and its target PHO2 was seen only at normal temperature (23°C). Flowering time ratio (16°C/23°C) revealed that miR399b-overexpressing plants and pho2 mutants showed increased sensitivity to ambient temperature changes. Expression analysis indicated that expression of TWIN SISTER OF FT (TSF) was increased in miR399b-overexpressing plants and pho2 mutants at 23°C, suggesting that their early flowering phenotype is associated with TSF upregulation. Taken together, our results suggest that miR399, an ambient temperature-responsive miRNA, plays a role in ambient temperature-responsive flowering in Arabidopsis.     

Regulatory network of microRNA399 and PHO2 by systemic signaling

Recently, we showed that microRNA399s (miR399s) control inorganic phosphate (Pi) homeostasis by regulating the expression of PHO2 encoding a ubiquitin-conjugating E2 enzyme 24. Arabidopsis (Arabidopsis thaliana) plants overexpressing miR399 or the pho2 mutant overaccumulate Pi in shoots. The association of Pi translocation and coexpression of miR399s and PHO2 in vascular tissues suggests their involvement in long-distance signaling. In this study, we used reciprocal grafting between wild-type and miR399-overexpressing transgenic plants to dissect the systemic roles of miR399 and PHO2. Arabidopsis rootstocks overexpressing miR399 showed high accumulation of Pi in the wild-type scions because of reduced PHO2 expression in the rootstocks. Although miR399 precursors or expression was not detected, we found a small but substantial amount of mature miR399 in the wild-type rootstocks grafted with transgenic scions, which indicates the movement of miR399 from shoots to roots. Suppression of PHO2 with miR399b or c was less efficient than that with miR399f. Of note, findings in grafted Arabidopsis were also discovered in grafted tobacco (Nicotiana benthamiana) plants. The analysis of the pho1 mutant provides additional support for systemic suppression of PHO2 by the movement of miR399 from Pi-depleted shoots to Pi-sufficient roots. We propose that the long-distance movement of miR399s from shoots to roots is crucial to enhance Pi uptake and translocation during the onset of Pi deficiency. Moreover, PHO2 small interfering RNAs mediated by the cleavage of miR399s may function to refine the suppression of PHO2. The regulation of miR399 and PHO2 via long-distance communication in response to Pi deficiency is discussed.     

Analysis of physiological and miRNA responses to Pi deficiency in alfalfa (<Emphasis Type="Italic">Medicago sativa</Emphasis> L.)

Key message

The induction of miR399 and miR398 and the inhibition of miR156, miR159, miR160, miR171, miR2111, and miR2643 were observed under Pi deficiency in alfalfa. The miRNA-mediated genes involved in basic metabolic process, root and shoot development, stress response and Pi uptake.

Abstract

Inorganic phosphate (Pi) deficiency is known to be a limiting factor in plant development and growth. However, the underlying miRNAs associated with the Pi deficiency-responsive mechanism in alfalfa are unclear. To elucidate the molecular mechanism at the miRNA level, we constructed four small RNA (sRNA) libraries from the roots and shoots of alfalfa grown under normal or Pi-deficient conditions. In the present study, alfalfa plants showed reductions in biomass, photosynthesis, and Pi content and increases in their root-to-shoot ratio and citric, malic, and succinic acid contents under Pi limitation. Sequencing results identified 47 and 44 differentially expressed miRNAs in the roots and shoots, respectively. Furthermore, 909 potential target genes were predicted, and some targets were validated by RLM-RACE assays. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed prominent enrichment in signal transducer activity, binding and basic metabolic pathways for carbohydrates, fatty acids and amino acids; cellular response to hormone stimulus and response to auxin pathways were also enriched. qPCR results verified that the differentially expressed miRNA profile was consistent with sequencing results, and putative target genes exhibited opposite expression patterns. In this study, the miRNAs associated with the response to Pi limitation in alfalfa were identified. In addition, there was an enrichment of miRNA-targeted genes involved in biological regulatory processes such as basic metabolic pathways, root and shoot development, stress response, Pi transportation and citric acid secretion.