The evolutionary-developmental analysis of plant microRNAs

Abstract

MicroRNAs (miRNAs) control many important aspects of plant development, suggesting these molecules may also have played key roles in the evolution of developmental processes in plants. However, evolutionary-developmental (evo-devo) studies of miRNAs have been held back by technical difficulties in gene identification. To help solve this problem, we have developed a two-step procedure for the efficient identification of miRNA genes in any plant species. As a test case, we have studied the evolution of the MIR164 family in the angiosperms. We have identified novel MIR164 genes in three species occupying key phylogenetic positions and used these, together with published sequence data, to partially reconstruct the evolution of the MIR164 family since the last common ancestor of the extant flowering plants. We use our evolutionary reconstruction to discuss potential roles for MIR164 genes in the evolution of leaf shape and carpel closure in the angiosperms. The techniques we describe may be applied to any miRNA family and should thus enable plant evo-devo to begin to investigate the contributions miRNAs have made to the evolution of plant development.

Figure 1.

Secondary structures and minimum free energy indices (MFEI) of pre-miR164s predicted from A. vulgaris (Avu-MIR164a–c), C. aquatica (Caq-MIR164a–c) and A. trichopoda (Atr-MIR164a).

Figure 2.

A phylogeny of the MIR164 family in the angiosperms. (a) A maximum likelihood phylogeny of the MIR164 family generated using the HKY model. Bootstrap supports are indicated. Accession numbers for the genes included are given in the electronic supplementary material, table S2. (b) The consensus view of angiosperm phylogeny (Stevens 2001), showing the relationships of species sampled in the present study.

Figure 3.

Semi-quantitative RT-PCR analysis of MIR164 gene expression. Amplifications of miR164 and GAPDH transcripts were simultaneously performed on first-strand cDNA prepared from the tissues indicated of: (a) A. thaliana, (b) A. vulgaris, (c) C. aquatica and (d) A. trichopoda. Genes corresponding to the B-clade, as identified in phylogenetic analyses, are indicated. Positive control amplifications from genomic DNA are also shown. Negative control amplifications, from RNA samples untreated with reverse transcriptase (not shown), yielded no PCR products in all cases.

Figure 4.

MIR164 evolution in the angiosperms. B-clade (red) and one or more other (blue) MIR164 genes were present in the last common ancestor of the extant angiosperms. Genes of both lineages show broadly conserved expression patterns between ANA grade angiosperms (A. trichopoda and C. aquatica) and eudicots (A. thaliana and A. vulgaris), as represented by plant organ symbols (flowers, leaves and roots).