Identification of miRNA precursors in the phloem of Cucurbita maxima.

Eugeny Tolstyko,Alexander Lezzhov,Andrey Solovyev

doi:10.7717/peerj.8269

Abstract

Plant development and responses to environmental cues largely depend on mobile signals including microRNAs (miRNAs) required for post-transcriptional silencing of specific genes. Short-range cell-to-cell transport of miRNA in developing tissues and organs is involved in transferring positional information essential for determining cell fate. Among other RNA species, miRNAs are found in the phloem sap. Long-distance transport of miRNA via the phloem takes a part in regulation of physiological responses to changing environmental conditions. As shown for regulation of inorganic phosphorus and sulfate homeostasis, mature miRNAs rather than miRNAs precursors are transported in the phloem as signaling molecules. Here, a bioinformatics analysis of transcriptomic data for Cucurbita maxima phloem exudate RNAs was carried out to elucidate whether miRNA precursors could also be present in the phloem. We demonstrated that the phloem transcriptome contained a subset of C. maxima pri-miRNAs that differed from a subset of pri-miRNA sequences abundant in a leaf transcriptome. Differential accumulation of pri-miRNA was confirmed by PCR analysis of C. maxima phloem exudate and leaf RNA samples. Therefore, the presented data indicate that a number of C. maxima pri-miRNAs are selectively recruited to the phloem translocation pathway. This conclusion was validated by inter-species grafting experiments, in which C. maxima pri-miR319a was found to be transported across the graft union via the phloem, confirming the presence of pri-miR319a in sieve elements and showing that phloem miRNA precursors could play a role in long-distance signaling in plants.

Highlights

In eukaryotes, microRNAs are 20–24 nucleotide-long RNA molecules that down-regulate gene expression by targeting mRNAs, which contain sequences complementary to miRNA, to either translational repression coupled with mRNA decay or endonucleolytic cleavage (Bartel, 2009)
To elucidate whether pre-miRNAs could be present in the phloem, we carried out a systematic analysis of transcriptomic data for Cucurbita maxima long phloem RNAs
We demonstrate that the phloem transcriptome contains a number of pri-miRNA sequences and that different pri-miRNA subsets are abundant in the phloem and leaf transcriptomes

Summary

INTRODUCTION

MicroRNAs (miRNAs) are 20–24 nucleotide-long RNA molecules that down-regulate gene expression by targeting mRNAs, which contain sequences complementary to miRNA, to either translational repression coupled with mRNA decay or endonucleolytic cleavage (Bartel, 2009). Unlike metazoans, mature plant miRNAs can be produced in the nucleus and exported into the cytoplasm in complex with AGO1 (Bologna et al, 2018), the silencing effector capable, as a part of RNA-induced silencing complex, of cleavage of target RNA molecules containing sites complementary to miRNAs (Baumberger & Baulcombe, 2005; Rogers & Chen, 2013). To elucidate whether pre-miRNAs could be present in the phloem, we carried out a systematic analysis of transcriptomic data for Cucurbita maxima long phloem RNAs. We demonstrate that the phloem transcriptome contains a number of pri-miRNA sequences and that different pri-miRNA subsets are abundant in the phloem and leaf transcriptomes. The presented data indicate that C. maxima pri-miRNAs can be selectively recruited to the phloem translocation pathway

MATERIALS AND METHODS

RESULTS AND DISCUSSION

CONCLUSION