Cloning of allene oxide cyclase gene from Leymus mollis and analysis of its expression in wheat-Leymus chromosome addition lines

Abstract

Leymus mollis (Triticeae; Poaceae) is a useful genetic resource for wheat (Triticum aestivum L.) breeding via wide hybridization to introduce its chromosomes and integrate its useful traits into wheat. Leymus mollis is highly tolerant to abiotic stresses such as drought and salinity and resistant to various diseases, but the genetic mechanisms controlling its physiological tolerance remain largely unexplored. We identified and cloned an allene oxide cyclase (AOC) gene from L. mollis that was strongly expressed under salt stress. AOC is involved in biosynthesis of jasmonic acid, an important signaling compound that mediates a wide range of adaptive responses. LmAOC cDNA consisted of 717 bp, coding for a protein with 238 amino acids that was highly similar to AOCs from barley (Hordeum vulgare) and other monocots. Subcellular localization using Nicotiana benthamiana confirmed it as a chloroplast-localized protein. LmAOC was found to be a multiple-copy gene, and that some copies were conserved and efficiently expressed in wheat-Leymus chromosome addition lines. LmAOC expression was upregulated under drought, heat, cold and wounding stresses, and by jasmonic acid and abscisic acid. Our results suggest that LmAOC plays an important role in L. mollis adaptation to abiotic stresses and it could be useful for wheat improvement.

Keywords: Leymus mollis; LmAOC; allene oxide cyclase; drought stress response; jasmonic acid; salinity; wheat.

Fig. 1

Alignment of the deduced amino acid sequence of LmAOC with other AOCs from different plant species. HvAOC, Hordeum vulgare (CAC83766.1); OsAOC, Oryza sativa (NP_001050447.1); SbAOC, Sorghum bicolor (XP_002465087.1); ZmAOC, Zea mays (ACG39242.1); AtAOC1, Arabidopsis thaliana (AT3G25760); AtAOC2, A. thaliana (AT3G25770); AtAOC3, A. thaliana (AT3G25780) and AtAOC4, A. thaliana (AT1G13280). Identical residues between all species are indicated by black letters on a dark gray background and conserved residues between more than three species are shown by black on light gray background.

Fig. 2

Subcellular localization of LmAOC in the chloroplast. LmAOC-GFP fusion protein transiently expressed in N. benthamiana leaves. (a–f) shows the localization in leave cells. (g–l) shows close view of the stomatal chloroplast. (a, g) green fluorescence of LmAOC-GFP; (b, i) red chloroplast autofluorescence; (c, k) merge between green fluorescence and the red chloroplast autofluorescence; (d, h) green fluorescence merged with bright field of the cells; (e, j) red chloroplast autofluorescence merged with bright field (f, l) bright field. Scale bars = 5 μm.

Fig. 3

Copy number of LmAOC in L. mollis, CS wheat and wheat-Leymus chromosome addition lines detected by southern blot analysis. DNA (10 μg) was digested with EcoRI, SalI and XhoI restriction enzymes, electrophoresed in 1.5% agarose gel and used for Southern blot. Bands corresponding to LmAOC copies derived from Leymus chromosomes are indicated with black arrow. Lm, L. mollis; CS, Chinese Spring; A, G or H, wheat-Leymus chromosome addition line harboring chromosome A, G or H from L. mollis, respectively.

Fig. 4

Northern blot analysis for LmAOC expression under some abiotic stresses. (A) salt stress; (B) drought stress; (C) JA, jasmonic acid treatment; (D) ABA, abscisic acid treatments. Total RNA (20 μg) isolated from control or treated plants was denatured in formaldehyde gel, transferred to Hybond-N⁺ nylon membranes and used for northern blot analysis. Plant subjected to either salt or drought were analyzed following day time course (0, 1 d, 2 d, 4 d and 6 d). Hormone treated plants were analyzed based on hours-day time course (0 h, 1 h, 3 h, 6 h, 1 d and 2 d).

Fig. 5

RT-PCR analysis for LmAOC expression under various abiotic stresses and hormone treatments. (A) salt stress; (B) drought stress; (C) heat stress; (D) cold stress; (E) wounding stress; (F) JA, jasmonic acid treatment; (G) ABA, abscisic acid treatment. First-strand cDNA was synthesized from one μg total RNA and used to perform RT-PCR using gene specific primers and action as internal control. Plants were analyzed following day time course (0, 1 d, 2 d, 3 d, 4 d, or 5 d) or hours-day time course (0 h, 1 h, 3 h, 6 h, 1 d and 1 d). (Top) Expression of LmAOC. (Bottom) Expression of actin used as internal control.

Fig. 6

RT-PCR analysis for LmAOC expression in CS wheat and wheat-Leymus chromosome addition lines under various abiotic stresses. One μg total RNA isolated from plants subjected to salt, drought, heat or cold stress was used for first-strand cDNA synthesis and RT-PCR. Cont, control plants, CS, Chinese Spring. A, G and H indicates wheat-Leymus chromosome addition lines harboring chromosome A, G or H from L. mollis, respectively. (Top) Expression of LmAOC. (Bottom) Expression of actin used as internal control.