A genome-wide study of the lipoxygenase gene families in Medicago truncatula and Medicago sativa reveals that MtLOX24 participates in the methyl jasmonate response

Abstract

Background: Lipoxygenase (LOX) is a multifunctional enzyme that is primarily related to plant organ growth and development, biotic and abiotic stress responses, and production of flavor-associated metabolites. In higher plants, the LOX family encompasses several isozymes with varying expression patterns between tissues and developmental stages. These affect processes including seed germination, seed storage, seedling growth, fruit ripening, and leaf senescence. LOX family genes have multiple functions in response to hormones such as methyl jasmonate (MeJA) and salicylic acid.

Results: In this study, we identified 30 and 95 LOX homologs in Medicago truncatula and Medicago sativa, respectively. These genes were characterized with analyses of their basic physical and chemical properties, structures, chromosomal distributions, and phylogenetic relationships to understand structural variations and their physical locations. Phylogenetic analysis was conducted for members of the three LOX subfamilies (9-LOX, type I 13-LOX, and type II 13-LOX) in Arabidopsis thaliana, Glycine max, M. truncatula, and M. sativa. Analysis of predicted promoter elements revealed several relevant cis-acting elements in MtLOX and MsLOX genes, including abscisic acid (ABA) response elements (ABREs), MeJA response elements (CGTCA-motifs), and antioxidant response elements (AREs). Cis-element data combined with transcriptomic data demonstrated that LOX gene family members in these species were most likely related to abiotic stress responses, hormone responses, and plant development. Gene expression patterns were confirmed via quantitative reverse transcription PCR. Several MtLOX genes (namely MtLOX15, MtLOX16, MtLOX20, and MtLOX24) belonging to the type I 13-LOX subfamily and other LOX genes (MtLOX7, MtLOX11, MsLOX23, MsLOX87, MsLOX90, and MsLOX94) showed significantly different expression levels in the flower tissue, suggesting roles in reproductive growth. Type I 13-LOXs (MtLOX16, MtLOX20, MtLOX21, MtLOX24, MsLOX57, MsLOX84, MsLOX85, and MsLOX94) and type II 13-LOXs (MtLOX5, MtLOX6, MtLOX9, MtLOX10, MsLOX18, MsLOX23, and MsLOX30) were MeJA-inducible and were predicted to function in the jasmonic acid signaling pathway. Furthermore, exogenous MtLOX24 expression in Arabidopsis verified that MtLOX24 was involved in MeJA responses, which may be related to insect-induced abiotic stress.

Conclusions: We identified six and four LOX genes specifically expressed in the flowers of M. truncatula and M. sativa, respectively. Eight and seven LOX genes were induced by MeJA in M. truncatula and M. sativa, and the LOX genes identified were mainly distributed in the type I and type II 13-LOX subfamilies. MtLOX24 was up-regulated at 8 h after MeJA induction, and exogenous expression in Arabidopsis demonstrated that MtLOX24 promoted resistance to MeJA-induced stress. This study provides valuable new information regarding the evolutionary history and functions of LOX genes in the genus Medicago.

Keywords: Arabidopsis thaliana; Lipoxygenase; Medicago; Methyl jasmonate; Overexpression.

Fig. 1

Phylogenetic analysis of LOX proteins in Arabidopsis thaliana, Glycine max, Medicago truncatula, and Medicago sativa. The phylogenetic tree was generated from six AtLOXs, 40 GmLOXs, 30 MtLOXs, and 111 MsLOXs. The branch colors represent LOX subgroups. The color and shape at the end of each branch represents the species: Arabidopsis (blue square), soybean (purple circle), M. truncatula (yellow five-pointed star), or M. sativa (green triangle)

Fig. 2

Gene structural analysis of Medicago truncatula LOXs. A Neighbor-joining phylogenetic tree showing relationships between MtLOX genes. B Motif analysis with the MEME suite yielded 10 motifs, each represented by a different color. C Conserved protein domain analysis with NCBI CDD predicted 12 conserved domains, each represented by a different color. D Exon–intron structural analysis. Yellow indicates exons, gray indicates introns, and green indicates untranslated regions (UTRs)

Fig. 3

Gene structural analysis of Medicago sativa LOXs. A Neighbor-joining phylogenetic tree showing relationships between MsLOX genes. B Motif analysis with the MEME suite yielded 10 motifs, each represented by a different color. C Conserved protein domain analysis with NCBI CDD predicted 12 conserved domains, each represented by a different color. D Exon–intron structural analysis. Yellow indicates exons, gray indicates introns, and green indicates untranslated regions (UTRs)

Fig. 4

Promoter analysis of LOX genes in Medicago truncatula and Medicago sativa. A The number of cis-acting elements in the 2-kb promoter region upstream of the translation start site of each MsLOX gene. B The number of cis-acting elements in the 2-kb promoter region upstream of the translation start site of each MtLOX gene. C Distribution of specific types of cis-acting elements in MtLOX and MsLOX genes

Fig. 5

Expression patterns of LOX genes in different tissues of Medicago truncatula and Medicago sativa. Relative expression levels of MtLOX (A) and MsLOX (B) genes in the leaves, flowers, and stems as determined with qRT-PCR. Expression levels of MtLOX and MsLOX genes were normalized to levels of MtActin and MsActin, respectively. Hierarchical gene clustering was performed with the normalization method “standard score” on log₂ transformed date. Colors indicates changes in expression compared to the leaf tissue, with red indicating an increase and blue indicating a decrease in expression

Fig. 6

Expression patterns of LOX genes in Medicago truncatula and Medicago sativa after exogenous methyl jasomate (MeJA) treatment. Relative expression levels of MtLOX (A) and MsLOX (B) genes at 0, 8, 12, and 48 h after treatment with 200 μM MeJA as determined with qRT-PCR. Expression levels of MtLOX and MsLOX genes were normalized to levels of MtActin and MsActin, respectively. Hierarchical gene clustering was performed with the normalization method “standard score” on log₂ transformed data. Colors indicate changes in expression compared to the 0 h timepoint, with red indicating an increase and blue indicating a decrease in expression

Fig. 7

Overexpression of MtLOX24 resulted in resistance to exogenous methyl jasmonate (MeJA) treatment of Arabidopsis seedlings. A Root growth among wild-type (WT) Arabidopsis seedlings and those overexpressing MtLOX24 (OE4 and OE5). Seven-d-old seedlings grown on ½ × Murashige and Skoog (MS) medium were transferred to ½ × MS medium supplemented with 0, 50, or 100 μM MeJA. Plants were assessed after two weeks. B Quantification of root length. C Fresh weight. D The number of lateral roots of Arabidopsis seedlings. Error bar represents the standard error calculated by three independent experiments

Fig. 8

Overexpression of the Medicago truncatula gene LOX24 in Arabidopsis thaliana resulted in resistance to exogenous methyl jasmonate (MeJA) treatment among seedlings grown in soil. A Phenotypes of wild-type (WT) and MtLOX24-overexpression (OE4 and OE5) Arabidopsis plants grown in soil with or without MeJA treatment for three weeks. B Quantification of chlorophyll content. C H₂O₂ content. D Relative conductivity in Arabidopsis seedlings. Error bars represent the standard error calculated from three independent experiments