Research Article: Identification and expression profiling of miRNAs in two color variants of carrot (Daucus carota L.) using deep sequencing

Date Published: March 7, 2019

Publisher: Public Library of Science

Author(s): Bhavana Bhan, Archana Koul, Deepak Sharma, Malik Muzafar Manzoor, Sanjana Kaul, Suphla Gupta, Manoj K. Dhar, Manoj Prasad.

http://doi.org/10.1371/journal.pone.0212746

Abstract

microRNAs represent small endogenous RNAs which are known to play a crucial role in various plant metabolic processes. Carrot being an important vegetable crop, represents one of the richest sources of carotenoids and anthocyanins. Most of the studies on microRNAs have been conducted in the aerial parts of the plants. However, carrot has the rare distinction of storing these compounds in roots. Therefore, carrot represents a good model system to unveil the regulatory roles of miRNAs in the underground edible part of the plant. For the first time, we report the genome wide identification and expression profiling of miRNAs in two contrasting color variants of carrot namely Orange Red and Purple Black using RNA-seq. Illumina sequencing resulted in the generation of 25.5M and 18.9M reads in Orange Red and Purple Black libraries, respectively. In total, 144 and 98 (read count >10), conserved microRNAs and 36 and 66 novel microRNAs were identified in Orange Red and Purple Black, respectively. Functional categorization and differential gene expression revealed the presence of several miRNA genes targeting various secondary metabolic pathways including carotenoid and anthocyanin biosynthetic pathways in the two libraries. 11 known and 2 novel microRNAs were further validated using Stem-Loop PCR and qRT-PCR. Also, target validation was performed for selected miRNA genes using RLM-RACE approach. The present work has laid a foundation towards understanding of various metabolic processes, particularly the color development in carrot. This information can be further employed in targeted gene expression for increasing the carotenoid and anthocyanin content in crop plants.

Partial Text

MicroRNAs (miRNAs) display a class of endogenous and non-coding small RNAs that play regulatory roles in modulating various metabolic processes in both plants and animals [1,2]. They act as trans-acting regulators, which modulate the gene expression at the post-transcriptional level and occasionally at the translational level. In plants, these ~21-nucleotide long RNAs are processed from the stem-loop regions of long primary transcripts by a Dicer-like enzyme [3]. Further, they are loaded into a silencing complex known as RNA induced silencing complex (RISC), where they direct the cleavage of complementary mRNAs/targets. The majority of plant miRNA target the encoded transcription factors or other regulatory molecules, such as proteins involved in ubiquitin and RNAi pathways.

Carrot has the rare distinction of storing carotenoids and anthocyanins in roots. These metabolites are synthesized by following two distinct metabolic pathways. In plants, most of the studies on both these metabolic pathways have been on the above ground parts; however, much remains to be explored about roots. Therefore, carrot represents an ideal system to understand the mechanism of carotenogenesis and biosynthesis of flavonoids in roots.

One of the essential aspects of small RNA regulated processes includes the elucidation of various mechanisms involved in color development. In the present study, differential gene expression of miRNAs was reported in two color variants of carrot. A total of 472 differentially expressed miRNAs were observed in Orange Red and Purple black carrot. Of these, 130 miRNAs were up regulated, 47 down regulated and 295 neutrally expressed. In terms of abundance, huge differences were observed among some miRNA families in the two varieties. 27 miRNAs in OR variety targeted both the carotenoid and flavonoid pathway, whereas the number in PB was 39. Most of the miRNAs in the carotenoid biosynthesis were upregulated in PB variety, which supports the high expression of carotenoid pathway genes in OR as compared to PB. Therefore, it is expected that these miRNAs might be playing a critical role in cultivar-specific color development processes. The combined miRNA and their target gene analysis suggested that differential expression of miRNAs in two color variants of carrot might be involved in the regulation of pigment biosynthesis and accumulation. Further, in-silico characterized and validated novel miRNAs might provide an important clue to regulate the color development in vegetable crops.

 

Source:

http://doi.org/10.1371/journal.pone.0212746

 

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