Identification of nutrient-responsive Arabidopsis and rapeseed microRNAs by comprehensive real-time polymerase chain reaction profiling and small RNA sequencing.

Comprehensive expression profiles of Arabidopsis (Arabidopsis thaliana) MIRNA genes and mature microRNAs (miRs) are currently not available. We established a quantitative real-time polymerase chain reaction platform that allows rapid and sensitive quantification of 177 Arabidopsis primary miR transcripts (pri-miRs). The platform was used to detect phosphorus (P) or nitrogen (N) status-responsive pri-miR species. Several pri-miR169 species as well as pri-miR398a were found to be repressed during N limitation, whereas during P limitation, pri-miR778, pri-miR827, and pri-miR399 species were induced and pri-miR398a was repressed. The corresponding responses of the biologically active, mature miRs were confirmed using specific stem-loop reverse transcription primer quantitative polymerase chain reaction assays and small RNA sequencing. Interestingly, the latter approach also revealed high abundance of some miR star strands. Bioinformatic analysis of small RNA sequences with a modified miRDeep algorithm led to the identification of the novel P limitation-induced miR2111, which is encoded by two loci in the Arabidopsis genome. Furthermore, miR2111, miR169, a miR827-like sequence, and the abundances of several miR star strands were found to be strongly dependent on P or N status in rapeseed (Brassica napus) phloem sap, flagging them as candidate systemic signals. Taken together, these results reveal the existence of complex small RNA-based regulatory networks mediating plant adaptation to mineral nutrient availability

Expression pattern suggests a role of MiR399 in the regulation of the cellular response to local Pi increase during arbuscular mycorrhizal symbiosis.

Many plants improve their phosphate (Pi) availability by forming mutualistic associations with arbuscular mycorrhizal (AM) fungi. Pi-repleted plants are much less colonized by AM fungi than Pi-depleted plants. This indicates a link between plant Pi signaling and AM development. MicroRNAs (miR) of the 399 family are systemic Pi-starvation signals important for maintenance of Pi homeostasis in Arabidopsis thaliana and might also qualify as signals regulating AM development in response to Pi availability. MiR399 could either represent the systemic low-Pi signal promoting or required for AM formation or they could act as counter players of systemic Pi-availability signals that suppress AM symbiosis. To test either of these assumptions, we analyzed the miR399 family in the AM-capable plant model Medicago truncatula and could experimentally confirm 10 novel MIR399 genes in this species. Pi-depleted plants showed increased expression of mature miR399 and multiple pri-miR399, and unexpectedly, levels of five of the 15 pri-miR399 species were higher in leaves of mycorrhizal plants than in leaves of nonmycorrhizal plants. Compared with nonmycorrhizal Pi-depleted roots, mycorrhizal roots of Pi-depleted M. truncatula and tobacco plants had increased Pi contents due to symbiotic Pi uptake but displayed higher mature miR399 levels. Expression levels of MtPho2 remained low and PHO2-dependent Pi-stress marker transcript levels remained high in these mycorrhizal roots. Hence, an AM symbiosis-related signal appears to increase miR399 expression and decrease PHO2 activity. MiR399 overexpression in tobacco suggested that miR399 alone is not sufficient to improve mycorrhizal colonization supporting the assumption that, in mycorrhizal roots, increased miR399 are necessary to keep the MtPho2 expression and activity low, which would otherwise increase in response to symbiotic Pi uptake