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

Laboratory of Molecular Biology, Wageningen University, 6700 ET Wageningen, The Netherlands.
Journal of Experimental Botany (Impact Factor: 5.53). 03/2013; 64(7). DOI: 10.1093/jxb/ert049
Source: PubMed


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.

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Available from: Rumyana Karlova, Jun 25, 2015
    • "In all vegetative and reproductive tissues tested, SlARF10A was upregulated significantly more than SlARF17 and SlARF10B, indicating that it serves as a major sly-miR160 target. In contrast to the miR160- targeted SlARFs, SlARF16A cleavage products were not recovered (Karlova et al., 2013) and accordingly our data indicated that its expression is not modified by sly-miR160 downregulation, strongly arguing against its sly-miR160-guided cleavage. It was shown that Arabidopsis miRNA-guided AGO1-RISC can block the recruitment or movement of ribosomes in target mRNAs by binding to sites with extensive complementarity in their coding sequence resulting in translational repression (Iwakawa and Tomari, 2013). "
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    ABSTRACT: Plant microRNAs play vital roles in auxin signaling via the negative regulation of Auxin Response Factors (ARFs). Studies have shown that targeting ARF10/16/17 by miR160 is indispensable for various aspects of development but its functions in the model crop tomato are unknown. Here we knockdown miR160 (sly-miR160) by Short Tandem Target Mimic (STTM160) and investigated its roles in tomato development. Northern blot analysis showed that miR160 is abundant in developing ovaries. In line with that, its downregulation perturbed ovary patterning as was evident by the excessive elongation of mutant ovaries proximal ends and thinning of their placenta. Following fertilization these morphological changes led to the formation of elongated, pear-shaped fruits reminiscent of those of the tomato ovate mutant. In addition, STTM160 expressing plants displayed abnormal floral organ abscission and produced leaves, sepals and petals with diminished blades indicating the requirement of sly-miR160 for these auxin mediated processes. We found that sly-miR160 depletion was always associated with the upregulation of SlARF10A, SlARF10B and SlARF17, of which the expression of SlARF10A increased the most. Despite its sly-miR160 legitimate site, SlARF16A mRNA levels did not change in response to sly-miR160 downregulation, suggesting that it may be regulated by an alternative mechanism other than mRNA cleavage. SlARF10A and SlARF17 were previously suggested to function as inhibiting ARFs. We propose that by adjusting the expression of a group of ARF repressors, of which SlARF10A is a primary target, sly-miR160 regulates auxin-mediated ovary patterning as well as floral organ abscission and lateral organ lamina outgrowth. This article is protected by copyright. All rights reserved.
    No preview · Article · Jan 2016 · The Plant Journal
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    • "Overexpression of miR396 in Arabidopsis enhanced drought tolerance (Liu et al., 2009). MicroRNAs in tomato have been primarily characterized in fruit development and ripening process (Moxon et al., 2008a; Mohorianu et al., 2011; Karlova et al., 2013; Din and Barozai, 2014). Recently Cao et al. (2014) profiled several cold-induced miRNAs in tomato. "
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    ABSTRACT: The 7B-1 tomato [Solarium lycopersicum L. 'Rutgers') is a male-sterile mutant with enhanced tolerance to abiotic stress in a blue-light (BL) specific manner compared with its wild-type (WT). This makes the 7B-1 a potential candidate for hybrid seed breeding and stress engineering. To identify small RNAs (sRNAs) linked to stress tolerance of 7B-1, two sRNA libraries from BL-grown 7B-1 and WT seedlings treated simultaneously with abscisic acid (ABA) and mannitol were sequenced, and sRNA profiles were compared. Twenty nine families of known microRNAs (miRNAs) and 27 putative novel miRNAs were identified from the two libraries. MiR5300, miR5301, miR2916, and a novel miRNA denoted miR#C were upregulated, while miR159, miR166, miR472, miR482, and two novel miRNAs, miR#A and miR#D, were downregulated in stress-treated 7B-1 seedlings. MiRNA targets with potential roles in stress regulation were validated by rapid amplification of 5' complementary DNA ends (5'-RACE) analysis. Expression of miR159, miR166, miR472, miR482, miR#A, and miR#D together with their targets were further investigated in response to ABA, mannitol, NaCI, and cold treatments and a strong negative correlation was observed between the levels of these miRNAs and expression of their targets. Only miR159 and miR166 responded to cold treatment. MiR#A and its target were regulated by ABA and mannitol as early as 0.5 h after the treatments, while other miRNAs and targets were regulated only after 2 h. This suggests a role in early response to stress for miR#A. Our data suggests that miR159, miR166, miR472, miR482, miR#A, and miR#D are likely to facilitate the BL-specific enhanced tolerance of 7B-1 to abiotic stress.
    Full-text · Article · Nov 2015 · The Plant Genome
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    • "For example, the target of miR156/157 is Colorless Non-Ripening (CNR) (Karlova et al., 2013), an epigenetic mutation of which could inhibit tomato fruit ripening (Manning et al., 2006). The target of miR172 is APETALA2 (AP2) (Karlova et al., 2013), which is a negative regulator of tomato fruit ripening (Chung et al., 2010; Karlova et al., 2011). In contrast, ripening-related lncRNAs have not been as comprehensively identified and functionally examined in tomato. "
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    ABSTRACT: Recently, long non-coding RNAs (lncRNAs) have been shown to play critical regulatory roles in model plants, such as Arabidopsis, rice, and maize. However, the presence of lncRNAs and how they function in fleshy fruit ripening are still largely unknown because fleshy fruit ripening is not present in the above model plants. Tomato is the model system for fruit ripening studies due to its dramatic ripening process. To investigate further the role of lncRNAs in fruit ripening, it is necessary and urgent to discover and identify novel lncRNAs and understand the function of lncRNAs in tomato fruit ripening. Here it is reported that 3679 lncRNAs were discovered from wild-type tomato and ripening mutant fruit. The lncRNAs are transcribed from all tomato chromosomes, 85.1% of which came from intergenic regions. Tomato lncRNAs are shorter and have fewer exons than protein-coding genes, a situation reminiscent of lncRNAs from other model plants. It was also observed that 490 lncRNAs were significantly up-regulated in ripening mutant fruits, and 187 lncRNAs were down-regulated, indicating that lncRNAs could be involved in the regulation of fruit ripening. In line with this, silencing of two novel tomato intergenic lncRNAs, lncRNA1459 and lncRNA1840, resulted in an obvious delay of ripening of wild-type fruit. Overall, the results indicated that lncRNAs might be essential regulators of tomato fruit ripening, which sheds new light on the regulation of fruit ripening.
    Full-text · Article · May 2015 · Journal of Experimental Botany
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