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Benzenoid biosynthesis in the flowers of Eriobotrya japonica: molecular cloning and functional characterization of p-methoxybenzoic acid carboxyl methyltransferase

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Abstract

Main conclusion

p -Methoxybenzoic acid carboxyl methyltransferase (MBMT) was isolated from loquat flowers. MBMT displayed high similarity to jasmonic acid carboxyl methyltransferases, but exhibited high catalytic activity to form methyl p -methoxybenzoate from p -methoxybenzoic acid.

Volatile benzenoids impart the characteristic fragrance of loquat (Eriobotrya japonica) flowers. Here, we report that loquat produces methyl p-methoxybenzoate, along with other benzenoids, as the flowers bloom. Although the adaxial side of flower petals is covered with hairy trichomes, the trichomes are not the site of volatile benzenoid formation. Here we identified four carboxyl methyltransferase (EjMT1 to EjMT4) genes from loquat and functionally characterized EjMT1 which we found to encode a p-methoxybenzoic acid carboxyl methyltransferase (MBMT); an enzyme capable of converting p-methoxybenzoic acid to methyl p-methoxybenzoate via methylation of the carboxyl group. We found that transcript levels of MBMT continually increased throughout the flower development with peak expression occurring in fully opened flowers. Recombinant MBMT protein expressed in Escherichia coli showed the highest substrate preference toward p-methoxybenzoic acid with an apparent K m value of 137.3 µM. In contrast to benzoic acid carboxyl methyltransferase (BAMT) and benzoic acid/salicylic acid carboxyl methyltransferase, MBMT also displayed activity towards both benzoic acid and jasmonic acid. Phylogenetic analysis revealed that loquat MBMT forms a monophyletic group with jasmonic acid carboxyl methyltransferases (JMTs) from other plant species. Our results suggest that plant enzymes with same BAMT activity have evolved independently.

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Abbreviations

SAMT:

Salicylic acid carboxyl methyltransferase

BAMT:

Benzoic acid carboxyl methyltransferase

BSMT:

Benzoic acid and salicylic acid carboxyl methyltransferase

JMT:

Jasmonic acid carboxyl methyltransferase

IAMT:

Indole-3-acetic acid (IAA) carboxyl methyltransferase

MBMT:

p-Methoxybenzoic acid carboxyl methyltransferase

SAM:

S-Adenosyl-l-methionine

RNA-Seq:

RNA-Sequencing

GC–MS:

Gas chromatography–mass spectrometry

qPCR:

Quantitative real-time polymerase chain reaction

SEM:

Scanning electron Microscope

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Acknowledgments

This research was partly supported by the Japan Society for the Promotion of Sciences KAKENHI (Grant Nos. 15K18690). Identification of volatile benzenoids was partly supported by the Development and Assessment of Sustainable Humanosphere system of the Research Institute for Sustainable Humanosphere, Kyoto University. We thank Mr. Tsutomu Hosouchi (Kazusa DNA Research Institute) for technical support in Illumina sequencing. We would also like to thank Ms. Caitlin Thireault for her proofreading of the manuscript. Radioisotope experiments in this work were performed in the Institute of Systems Biology and Radioisotope Analysis, Science Research Center, Yamaguchi University.

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Authors and Affiliations

  1. Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi, 753-8515, Japan

    Takao Koeduka, Mami Kajiyama & Kenji Matsui

  2. Department of Research and Development, Kazusa DNA Research Institute, Chiba, 292-0818, Japan

    Hideyuki Suzuki

  3. Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto, 611-0011, Japan

    Takumi Furuta & Tomohiko Tsuge

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  1. Takao Koeduka
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Correspondence to Takao Koeduka.

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Table S1. Oligonucleotide sequences used for the semi-quantitative RT-PCR of EjMTs and EjACT (DOCX 71 kb)

425_2016_2542_MOESM2_ESM.pptx

Figure S1. GC–MS analysis of volatile compounds extracted from loquat flowers. Volatile compounds in the different developmental stages: leaves, flower stage 1, flower stage 2, flower stage 3 were analyzed by GC–MS and the peaks identified as volatile benzenoid are numbered (a). Chemical structures detected in panel (a) are shown (b) (PPTX 526 kb)

425_2016_2542_MOESM3_ESM.pptx

Figure S2. Effect of abrasive treatment to remove hairy trichomes on content of the major volatile benzenoids in loquat flowers. (a) Upper and lower panels show the detached petals before and after the abrasive treatment to remove trichomes, respectively. (b) Black bar, non-treated flower petals; white bar, flower petals from which hairy trichomes had been removed; gray bar, flower petals from which hairy trichomes had been removed and dipped in organic solvent. n.d. indicates not detected. Asterisks show significant difference (ANOVA, *P < 0.05, **P < 0.01). (c) GC–MS analysis of volatile benzenoids extracted from whole petals and hairy trichomes. The number of peaks indicates the chemical structures as shown in Figure S1 (PPTX 491 kb)

425_2016_2542_MOESM4_ESM.pptx

Figure S3. GC–MS analysis of the reaction product obtained in EjMBMT assays with p-methoxybenzoic acid. (a) Proposed enzymatic conversion of p-methoxybenzoic acid and S-adenosyl-L-Met (SAM) to methyl p-methoxybenzoate by EjMBMT enzyme activity. (b) The purified EjMBMT, before and after boiling, were incubated with 0.5 mM p-methoxybenzoic acid (peak 1) and 0.5 mM SAM. Hexane extracts of the reactions were analyzed by GC–MS and a single reaction product, methyl p-methoxybenzoate, was detected as peak 2 (PPTX 161 kb)

425_2016_2542_MOESM5_ESM.pptx

Figure S4. Alignments around the important residues determining methylating activity for jasmonic acid and salicylic acid/benzoic acid. Key residues (Ser and Tyr) are indicated by bold (PPTX 64 kb)

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Koeduka, T., Kajiyama, M., Suzuki, H. et al. Benzenoid biosynthesis in the flowers of Eriobotrya japonica: molecular cloning and functional characterization of p-methoxybenzoic acid carboxyl methyltransferase. Planta 244, 725–736 (2016). https://doi.org/10.1007/s00425-016-2542-2

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