Identification, Phylogeny, and Comparative Expression of the Lipoxygenase Gene Family of the Aquatic Duckweed, Spirodela polyrhiza, during Growth and in Response to Methyl Jasmonate and Salt

Abstract

Lipoxygenases (LOXs) (EC 1.13.11.12) catalyze the oxygenation of fatty acids and produce oxylipins, including the plant hormone jasmonic acid (JA) and its methyl ester, methyl jasmonate (MeJA). Little information is available about the LOX gene family in aquatic plants. We identified a novel LOX gene family comprising nine LOX genes in the aquatic plant Spirodela polyrhiza (greater duckweed). The reduced anatomy of S. polyrhiza did not lead to a reduction in LOX family genes. The 13-LOX subfamily, with seven genes, predominates, while the 9-LOX subfamily is reduced to two genes, an opposite trend from known LOX families of other plant species. As the 13-LOX subfamily is associated with the synthesis of JA/MeJA, its predominance in the Spirodela genome raises the possibility of a higher requirement for the hormone in the aquatic plant. JA-/MeJA-based feedback regulation during culture aging as well as the induction of LOX gene family members within 6 h of salt exposure are demonstrated.

Keywords: MeJA; Spirodela polyrhiza; duckweed; lipoxygenases (LOXs); oxylipin; phylogenetics; salt.

Figure 1

Molecular phylogenetic relationships of the Spirodela polyrhiza lipoxygenase (LOX) family with LOX gene families from additional plants. The evolutionary history was inferred using the maximum likelihood method based on the JTT matrix-based model. The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analyze. The analysis involved 63 amino acid sequences from five different plants: duckweed (Sp), tomato (Sl), Arabidopsis (At), rice (Os), and poplar (Pt). All positions with less than 95% site coverage were eliminated. Evolutionary analyses were conducted in MEGA7. The bootstrap values of the confidence levels are shown as percentages at branch nodes. The LOX gene family of each species is color coded. LOXs of different species fall into two separate groups: 9-LOX and 13-LOX. The 13-LOX group was further subdivided into type I and type II. Phylogenetic analysis assigned seven LOX proteins from Spirodela to the 13-LOX group and two LOX proteins to the 9-LOX group.

Figure 2

Identification of conserved histidine (H) residues in the 38 aa signature LOX motif in Spirodela. (A) 38-residue motif among Spirodela LOX sequences. The sequence logo was created with the indigenous nine LOX protein sequences. The average height of each stack indicates the sequence conservation at that position and the height of each residue letter indicates the relative distribution frequency of the corresponding amino acid residue in the 38 amino acid long motif. (B) Sequence alignment of the 38-residue long motif in the Spirodela LOX proteins. The conserved histidine residues are highlighted as bold H.

Figure 3

Spirodela LOX family genes’ relatedness and intron-exon arrangements. The evolutionary history was inferred using the maximum likelihood method based on the JTT matrix-based model. The percentage of trees in which the associated taxa clustered together is shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved nine amino acid sequences. All positions with less than 95% site coverage were eliminated. Evolutionary analyses were conducted in MEGA7 [51]. The Spirodela LOX subfamilies 9-LOX and 13-LOX are designated, with 13-LOX further separated into type I (13-LOX- I) and type II (13-LOX- II). Schematic genomic organization for each LOX gene was generated using the Gene Structure Display Server (GSDS 2.0; http://gsds.cbi.pku.edu.cn/). Exons (CDS) and introns are represented by blue boxes and black dashed lines, respectively. The sizes of exons and introns are proportional to their sequence lengths.

Figure 4

Spirodela LOX sequence identities. (A) Coding DNA sequence and (B) protein sequence identity matrices were generated using EMBOSS stretcher (https://www.ebi.ac.uk/Tools/psa/emboss_stretcher/). The values in bold face are discussed in the text.

Figure 5

qRT-PCR analysis of LOX genes in two clones of Spirodela polyrhiza, Sp7498and Sp7003. Cultures were grown for 28 days in nutrient solution and samples collected at 14, 21, and 28 days of growth. Confluence of plants in the culture flasks was reached after 21 days of growth. mRNA expression profiles of whole plants from the two clones were analyzed. SpACT and Sp18SrRNA housekeeping genes were used to normalize the expression of LOX genes, as described in Materials and Methods. Analysis of variance (ANOVA) with Dunnett’s multiple comparisons test was performed for significant differences in LOX gene expression in the aging plants. Statistical significance between expression data points was assessed against the 14-day expression profiles and categorized as * p < 0.05, ** p < 0.01, and *** p < 0.001 using Graph Pad (version 8.0); ns: not significant.

Figure 6

Comparative qRT-PCR analysis of LOX genes in Spirodela polyrhiza 7498, Lemna minor 8627, and Landoltia punctata 5562. Plants were taken for expression analysis at different ages of culture. All cultures were grown for 28 days in nutrient solution and samples were collected at 14, 21, and 28 days of growth. The SpACT and Sp18SrRNA housekeeping genes were used to normalize the expression of LOX genes, as described in Materials and Methods. (A) LOX1, (B) LOX3, (C) LOX4, (D) LOX6, and (E) LOX7 expression comparison was made among the three strains. ANOVA with Dunnett’s multiple comparisons test was performed for significant differences in LOX gene expression in the aging tissues among the duckweed species. Statistical significance between aging expression data points was assessed against the 14-day expression profiles and categorized as ** p < 0.01, using Graph Pad (version 8.0); ns: not significant.

Figure 7

Methyl jasmonate (MeJA) induced feed-back regulation of Spirodela LOX gene family. MeJA (10 μM) was added to 14-day-old S. polyrhiza 7498 cultures, as previously described [35]. Samples were collected at 0, 1, 3, 6, and 12 h after treatment. Gene expression data were analyzed in treated and untreated fronds by qRT-PCR. Statistical significance between treatment data points was assessed with respect to control for each time point and categorized as * p < 0.05, ** p < 0.01, and **** p < 0.0001 using graph pad (version 8.0); ns: not significant.

Figure 8

Comparative qRT-PCR analysis of LOX genes in Spirodela in response to exogenous salt. Five fronds of S. polyrhiza 7498 were grown for 14 days; then 200 mM NaCl solution was added; and samples were harvested at 0, 1, 3, 6, and 12 h [36]. Gene expression data were analyzed by qRT-PCR. The experiment was repeated three times (n = 3) with each sample size of approximately 100 fronds. Analysis of variance (ANOVA) with Dunnett’s multiple comparisons test was performed for significant differences. Statistical significance between data points was assessed against 0 h versus other time points of expression profiles using graph pad (version 8.0).