Novel and mechanical stress-responsive MicroRNAs in Populus trichocarpa that are absent from Arabidopsis

Abstract

MicroRNAs (miRNAs) are small, noncoding RNAs that can play crucial regulatory roles in eukaryotes by targeting mRNAs for silencing. To test whether miRNAs play roles in the regulation of wood development in tree species, we isolated small RNAs from the developing xylem of Populus trichocarpa stems and cloned 22 miRNAs. They are the founding members of 21 miRNA gene families for 48 miRNA sequences, represented by 98 loci in the Populus genome. A majority of these miRNAs were predicted to target developmental- and stress/defense-related genes and possible functions associated with the biosynthesis of cell wall metabolites. Of the 21 P. trichocarpa miRNA families, 11 have sequence conservation in Arabidopsis thaliana but exhibited species-specific developmental expression patterns, suggesting that even conserved miRNAs may have different regulatory roles in different species. Most unexpectedly, the remaining 10 miRNAs, for which 17 predicted targets were experimentally validated in vivo, are absent from the Arabidopsis genome, suggesting possible roles in tree-specific processes. In fact, the expression of a majority of the cloned miRNAs was upregulated or downregulated in woody stems in a manner consistent with tree-specific corrective growth against tension and compression stresses, two constant mechanical loads in trees. Our results show that plant miRNAs can be induced by mechanical stress and may function in one of the most critical defense systems for structural and mechanical fitness.

Figure 1.

Predicted Stem-Loop Structures of Precursors Containing the Newly Cloned miRNA Sequence (Red).

Figure 2.

Developmental and Mechanical Stress–Responsive Expression of Populus miRNAs. (A) RNA gel blots of total RNA isolated from leaf (L), phloem (P), developing xylem (X), tension-stressed developing xylem (Xtw), and compression-stressed developing xylem (Xow) were probed with end-labeled oligonucleotides. The 5S rRNA bands were visualized by ethidium bromide staining of polyacrylamide gels and served as loading controls. nt, nucleotides. (B) Relative expression of miRNAs in leaf (L), phloem (P), developing xylem (X), tension-stressed developing xylem (Xtw), and compression-stressed developing xylem (Xow) was analyzed by real-time PCR. The 5.8S rRNA was selected as a reference. The data are means of three measurements ± se.

Figure 3.

Experimental Validation of the Predicted mRNA Targets for the Newly Cloned ptr-miR408, 473a, 474a, 475a, and 477a. The mRNA cleavage sites were determined by modified 5′ RNA ligase-mediated RACE. Heavy black lines represent ORFs. The lines flanking gray regions represent nontranslated regions (if known) and miRNA complementary sites, with the nucleotide positions within the ORF indicated. The mRNA sequence of each complementary site from 5′ to 3′ and the cloned miRNA sequence (bold) from 3′ to 5′ are shown in the expanded regions. Watson-Crick pairing (vertical dashes) and G:U wobble pairing (circles) are indicated. Vertical arrows indicate the 5′ termini of miRNA-guided cleavage products, as identified by 5′-RACE, with the frequency of clones shown. Positions of the nesting and the nested primers used for 5′-RACE are indicated by horizontal arrows. For targets eugene3.03440011, eugene3.00700255, and eugene3.08910001 predicted for ptr-miR474a, 5′-RACE was performed using the same set of primers, yielding products with the identical sequence for which origin could not be certain. Therefore, at least one of them is an authentic target for ptr-miR474a.

Figure 4.

Experimental Validation of the Predicted mRNA Targets for the Newly Cloned ptr-miR475a, 476a, 478a, 479, 480a, and 482. The ptr-miR475a targets shown in this figure and in Figure 3 were validated using the same set of degenerate primers. The partial flanking sequence of ptr-miR482 in the precursor is also shown.