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Regulation of barley miRNAs upon dehydration stress correlated with target gene expression

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Abstract

We aim to identify conserved and dehydration responsive microRNAs (miRNAs) in Hordeum vulgare (barley). A total of 28 new barley miRNAs belonging to 18 distinct miRNA families were identified. Detailed nucleotide analyses revealed that barley pre-miRNAs are in the range of 46–114 nucleotides with average of 77.14. Using 28 newly detected miRNAs as queries, 445 potential target mRNAs were predicted. The predicted miRNAs were differentially expressed and some of them behaved similarly in leaf and root tissues upon stress treatment. Hvu-MIR156, Hvu-MIR166, Hvu-MIR171, and Hvu-MIR408 were detected as dehydration stress-responsive barley miRNAs. To discover target transcripts of barley miRNAs a modified 5′ RLM-RACE was performed and seven cleaved miRNA transcripts were retrieved from drought stressed leaf samples. In silico analysis indicated 15 potential EST targets. Measurement of expression levels showed a positive correlation between levels of miRNA expression and suppression of their target mRNA transcripts in dehydration-stress-treated barley.

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Abbreviations

ΔG:

Folding free energies

AGO1:

Argonaute-1

ARF:

Auxin response transcription factor

DCL1:

Dicer-like 1

GSS:

Genome survey sequence

miRNA:

Micro-RNA

RT:

Real time

EST:

Expressed sequence tag

EREBPs:

Ethylene-responsive element binding proteins

MFE:

Minimal folding free energy

MFEI:

Minimal folding free energy index

miRNA*:

miRNA star strand

NCBI:

National center for biotechnology information

nt:

Nucleotide(s)

pre-miRNA:

MicroRNA precursor

pri-miRNAs:

MicroRNA primary

RISC:

RNA-induced silencing complex

5′ RLM-RACE:

RNA ligase-mediated 5′ rapid amplification of cDNA ends

qRT-PCR:

Quantitative real-time PCR

SBP:

Squamosa promoter binding proteins

SCL6:

Scarecrow-like transcription factor 6

Ser:

Serine

SPL:

SBP-like proteins

Thr:

Threonine

ORF:

Open reading frame

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Authors and Affiliations

  1. Biological Sciences and Bioengineering Program, Faculty of Engineering and Natural Sciences, Sabanci University, Istanbul, Turkey

    Melda Kantar, Turgay Unver & Hikmet Budak

Authors
  1. Melda Kantar
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  2. Turgay Unver
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  3. Hikmet Budak
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Corresponding author

Correspondence to Hikmet Budak.

Additional information

Melda Kantar and Turgay Unver should be regarded as joint first authors.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Table 1

Pre-miRNA sequences of predicted barley miRNAs (DOC 66 kb)

Supplementary Table 2

Computationally identified target ESTs for H. vulgare miRNAs (DOC 45 kb)

Supplementary Table 3

List of primers used for quantification and validation of computer-based H. vulgare miRNAs (DOC 38 kb)

Supplementary Table 4

In silico found miRNA targets that were intended to be amplified in RLM-RACE reactions. The green part indicates in silico miRNA cleavage site, yellow parts indicate reverse primers (DOC 25 kb)

Supplementary Table 5

miRNA cleaved mRNA sequences retrieved with RLM-RACE experiments (DOC 32 kb)

Supplementary Table 6

Mean quantification values and corresponding standard deviations of triplicates for miRNA and target real-time PCRs (DOC 90 kb)

Supplementary Figure 1

Sequence alignment of RACE sequences with computationally proposed Hvu-miRNAs (DOC 27 kb)

Supplementary Figure 2

Sequence alignment of RACE sequences with known miRNAs from different plant species. * indicates reverse complimentary miRNA (DOC 26 kb)

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Kantar, M., Unver, T. & Budak, H. Regulation of barley miRNAs upon dehydration stress correlated with target gene expression. Funct Integr Genomics 10, 493–507 (2010). https://doi.org/10.1007/s10142-010-0181-4

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  • DOI: https://doi.org/10.1007/s10142-010-0181-4

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