Melon13-lipoxygenase CmLOX18 may be involved in C6 volatiles biosynthesis in fruit

Abstract

To better understand the function role of the melon CmLOX18 gene in the biosynthesis of C6 volatiles during fruit ripening, we biochemically characterized CmLOX18 and identified its subcellular localization in transgenic tomato plants. Heterologous expression in yeast cells showed that the molecular weight of the CmLOX18 protein was identical to that predicted, and that this enzyme possesseed lipoxygenase activity. Linoleic acid was demonstrated to be the preferred substrate for the purified recombinant CmLOX18 protein, which exhibited optimal catalytic activity at pH 4.5 and 30 °C. Chromatogram analysis of the reaction product indicated that the CmLOX18 protein exhibited positional specificity, as evidenced by its release of only a C-13 oxidized product. Subcellular localization analysis by transient expression in Arabidopsis protoplasts showed that CmLOX18 was localized to non-chloroplast organelles. When the CmLOX18 gene was transgenically expressed in tomato via Agrobacterium tumefaciens-mediated transformation, it was shown to enhance expression levels of the tomato hydroperoxide lyase gene LeHPL, whereas the expression levels of six TomLox genes were little changed. Furthermore, transgenic tomato fruits exhibited increases in the content of the C6 volatiles, namely hexanal, (Z)-3-hexanal, and (Z)-3-hexen-1-ol, indicating that CmLOX18 probably plays an important role in the synthesis of C6 compounds in fruits.

Figure 1

Analysis of recombinant CmLOX18 (a) Western blot analysis. Total proteins (20 μg) were isolated from yeast cells expressing CmLXO18 which was harvested at 24 hours induction and separated on 12% SDS/PAGE gel, then transferred to a PVD membrane and LOX detected with anti-His antibody. (b) SDS-PAGE analysis of the recombinant CmLOX18. The proteins were stained with Coomassie brilliant blue R-250. M: Positions of marker protein. 1: SDS-PAGE of the purified CmLOX18 extracted from pYES2/lox transformed yeast cultures (20 μg). The expressed CmLOX18 was indicated by red arrows.

Figure 2

The optimum temperature (a) and Ph (b) for recombinant CmLOX18. The effects of pH and temperature on the enzymatic activity of recombinant CmLOX18 were determined using linoleic acid (LA) as substrates. The recombinant CmLOX18 proteins displayed the highest catalytic activity at pH 4.5 (a) respectively. The optimum temperatures for CmLOX18 were observed at 30 °C (b), respectively. The maximum activity was estimated as 100%. Means ± SD were obtained from three independent measurements.

Figure 3

Determination of positional specificity of the recombinant CmLOX18. (a) The retention time of the isomers produced by soybean LOX1 compared with authentic standards of 13-HPOD and 9-HPOD. (b) SP-HPLC analysis of the reaction mixture catalysed by recombinant CmLOX18. Boxes: chiral-phase HPLC showing the enantiomer composition of 13-HPOD.

Figure 4

Subcellular localization of oriental melon CmLOX18 in Arabidopsis protoplasts. Arabidopsis protoplasts were transformed by the vectors containing the 35S-GFP (A) and 35S-CmLOX18-GFP (B), respectively. Chlorophyll and GFP fluorescence were examined using Zeiss LSM510 confocal microscope excited with a 488-nm Ar laser line. Merge is the computed overlay of the two fluorescence images. Reference bar is 5 μm.

Figure 5

Molecular analysis of T0 transegenic tomato liners expressing CmLOX18. (a) Southern blot of wild-type control and four T0 transgenic tomato lines expression CmLOX18. Genomic DNA was prepared from young leaf material from wild-type control and transgenic plants: 1, 2, and 3 and V (35S-overexpression CmLOX18 construct). The genomic DNA (10 μg/lane) was digested with HindШ and separated in a 0.8% (w/v) agarose gel. Blotted DNA was hybridized to a probe prepared from the bar gene. (b) Detection of CmLOX18 cDNA by PCR analysis. Genomic DNA was extracted from transgenic lines. (c) Western blot. Proteins were extracted and Western blot was carried according to standard procedures using anti-e GFP Mouse Monoclonal antibody. (d) Images of wild-type control and twoT0 transgenic tomato leaves.

Figure 6

Transcript levels of CmLOX18 and LeHPL in wild type (“Zhongshu 6”) and transgenic (CM18-01, 03 and 04) ripe fruits. Transcripts were quantified by Quantitative PCR. Values are mean ± standard error of three replicates. Significant differences are indicated with asterisks above the bars: *P ≤ 0.05, **P ≤ 0.01.

Figure 7

Transcript levels of TomLOXA-F in wild type (“Zhongshu 6”) and transgenic (CM18-01, 03 and 04) ripe fruits. Transcripts were quantified by Quantitative PCR. Values are mean ± standard error of three replicates. Significant differences are indicated with asterisks above the bars: *P ≤ 0.05, **P ≤ 0.01.

Figure 8

Overexpression CmLOX18 in fruits C6 and C5 volatiles emission in wild type (“Zhongshu 6”) and transgenic (CM18-01, 03 and 04) ripe fruits. Values are mean ± standard error of three replicates. Significant differences are indicated with asterisks above the bars: *P ≤ 0.05, **P ≤ 0.01.