Identification of microRNA targets in tomato fruit development using high-throughput sequencing and degradome analysis

Abstract

MicroRNAs (miRNAs) play important roles in plant development through regulation of gene expression by mRNA degradation or translational inhibition. Despite the fact that tomato (Solanum lycopersicum) is the model system for studying fleshy fruit development and ripening, only a few experimentally proven miRNA targets are known, and the role of miRNA action in these processes remains largely unknown. Here, by using parallel analysis of RNA ends (PARE) for global identification of miRNA targets and comparing four different stages of tomato fruit development, a total of 119 target genes of miRNAs were identified. Of these, 106 appeared to be new targets. A large part of the identified targets (56) coded for transcription factors. Auxin response factors, as well as two known ripening regulators, colorless non-ripening (CNR) and APETALA2a (SlAP2a), with developmentally regulated degradation patterns were identified. The levels of the intact messenger of both CNR and AP2a are actively modulated during ripening, by miR156/157 and miR172, respectively. Additionally, two TAS3-mRNA loci were identified as targets of miR390. Other targets such as Argonaute 1 (AGO1), shown to be involved in miRNA biogenesis in other plant species, were identified, which suggests a feedback loop regulation of this process. In this study, it is shown that miRNA-guided cleavage of mRNAs is likely to play an important role in tomato fruit development and ripening.

Fig. 1.

Target plots of validated target mRNAs. The abundance of each sequenced read is plotted as a function of the position of its 5’ end in the transcript. In red are the peaks of signatures at the predicted cleavage site of the corresponding miRNAs (arrows). (A) Cleavage features in SlAP2a mRNA by miR172a from the breaker (Br) fruit stage degradome library. (B) Cleavage features in CNR mRNA by miR156a at the Br fruit stage. (C) Cleavage features in SlDRM5 mRNA by miR396 at the Br fruit stage. (D) RLM-RACE confirmation of the SlDRM5 cleavage site by miR396. The numbers above the vertical arrow indicate the number of sequences found at the exact miR396 cleavage site.

Fig. 2.

miRNA-directed cleavage product abundance at different fruit stages. Normalized sequenced read abundance [peak height=tags per 10 million (TP10M)] on the y-axis. (A) Cleavage product abundances of AGO1-like (Solyc02g069260.2.1) by miR403 and of AGO1-like (Solyc06g072300.2.1) by miR168. (B) Cleavage product abundances of GAMYB-like 1 and of GAMYB-like 2 by miR159. (C) Cleavage product abundances of SlTIR1, and of two other TIR1-like mRNAs by miR393. (D) Cleavage product abundance of AP2a by miR172. (E) Cleavage product abundance of CNR by miR156.

Fig. 3.

Relative level of degradation of AP2a and of CNR mRNA by miR172 and miR156, respectively. Relative expression profiles of AP2a and CNR obtained by quantitative reverse transcription PCR with two different primers sets: in grey, primers spanning the miRNA cleavage site (detecting the level of the non-cleaved mRNA); in black, primers for the 3’ UTR, downstream of the cleavage sites (detecting both the intact and the cleaved mRNA) in tomato wild-type fruits at the 5 DAP, MG, Br, and RR stage. Error bars represent the SE. The expression levels are normalized relative to 5 DAP, which was set at 1, for each gene separately. (A) Relative expression profile of AP2a. (B) Relative expression profile of CNR. (C) Model of tomato fruit ripening control by CNR, AP2a, miR156, and miR172. The transcription factor genes AP2a and CNR are required for the normal ripening process. AP2a has a negative regulatory function in ethylene synthesis besides its positive ripening regulatory functions. CNR and AP2a regulate each other’s expression in opposite ways and their expression is also negatively regulated by miR156 and miR172, respectively.