Natural antisense transcripts of Trifolium repens dehydrins

Dehydrins (group 2 LEA proteins) tend to accumulate late in embryogenesis and in response to stress leading to cell dehydration (e.g., drought, low temperature, and salinity). The expression pattern of group 2 LEA genes is frequently associated with higher tolerance of crop plants to abiotic stresses such as cold and drought. All dehydrins have at least one conserved, lysine-rich 15-amino acid domain, EKKGIMDKIK EKLPG, named the K-segment near the C-terminus, and some of them may have a track of Ser residues (the S-segment), and/or a consensus motif (in one or more copies), T/VDEYGNP (Y-segment) located near the N-terminus. The number and order of the Y-, S-, and K-segments define different dehydrin sub-classes: Y n SK n , Y n K n , SK n , K n and K n S. Detailed analyses of promoter regions of some DHN genes provided evidence for a close relationship between dehydrin expression patterns and the various upstream and downstream cis-regulatory elements present in the sequences. Published data outlined that the regulation of expression of some dehydrin genes is elaborate and could be a result of several interacting factors. Results from a recent study aimed at the identification of different dehydrin types in white clover (Trifolium repens) demonstrated the complex nature of dehydrin-coding sequences, which may lead to a high variability among the transcripts originating from a single gene. The aim of the present study was to confirm experimentally the existence of previously predicted dehydrin NAT transcripts and to compare their abundance in white clover grown under different abiotic stress treatments. Natural antisense RNAs could potentially regulate the expression of their sense partner(s) at either transcriptional or post-transcriptional level. A recent work described that a rice cis-natural antisense RNA acts as a translational enhancer for its cognate mRNA. SAS pairs in plants have the potential to become substrates for the ribonuclease III-like enzyme Dicer to produce short interfering RNAs (siRNAs) and natural antisense microRNAs (nat-miRNAs) with regulatory potential. Many studies have confirmed that abiotic or biotic stresses induce production of the so-called nat-siRNA (natural-small interfering RNA) from cis-NATs. Plants used in the present study (Trifolium repens, cv Apis) were grown hydroponically on a standard nutrient solution for 2 weeks (23 °C night / 26 °C day; 80% relative humidity; 14 h photoperiod; photosynthetic active irradiation of 200 μmol m s). Some of them were subjected to 72 h treatment with PEG (100 g PEG-6000 added to 1 L nutrient solution), and others were transferred on standard nutrient medium supplemented with 1 μM ABA or 75 mM NaCl and were grown for additional 14 d. Total RNA extracted from differently treated plants was reversely transcribed in sense and in antisense direction, with oligo(dT 23 ) (Sigma-Aldrich) and forward gene specific primers (Y n K n F: ATGAATATGG AAACCCAGTG