Abstract
Main conclusion
An Arabidopsis S-adenosyl-l-methionine-dependent methyltransferase belonging to the SABATH family catalyzes the specific carboxymethylation of (11R)-carlactonoic acid.
Abstract
Methyl carlactonoate (MeCLA), found in Arabidopsis (Arabidopsis thaliana) as a non-canonical strigolactone (SL), may be a biosynthetic intermediate of various non-canonical SLs and biologically active as a plant hormone. MeCLA is formed from carlactonoic acid (CLA), but the methyltransferases (MTs) converting CLA to MeCLA remain unclear. Previous studies have demonstrated that the carboxymethylation of acidic plant hormones is catalyzed by the same protein family, the SABATH family (Wang et al. in Evol Bioinform 15:117693431986086. https://doi.org/10.1177/1176934319860864, 2019). In the present study, we focused on the At4g36470 gene, an Arabidopsis SABATH MT gene co-expressed with the MAX1 gene responsible for CLA formation for biochemical characterization. The recombinant At4g36470 protein expressed in Escherichia coli exhibited exclusive activity against naturally occurring (11R)-CLA among the substrates, including CLA enantiomers and a variety of acidic plant hormones. The apparent Km value for (11R)-CLA was 1.46 μM, which was relatively smaller than that of the other Arabidopsis SABATH MTs responsible for the carboxymethylation of acidic plant hormones. The strict substrate specificity and high affinity of At4g36470 suggested it is an (11R)-CLA MT. We also confirmed the function of the identified gene by reconstructing MeCLA biosynthesis using transient expression in Nicotiana benthamiana. Phylogenetic analysis demonstrated that At4g36470 and its orthologs in non-canonical SL-producing plants cluster together in an exclusive clade, suggesting that the SABATH MTs of this clade may be involved in the carboxymethylation of CLA and the biosynthesis of non-canonical SLs.
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Data availability
The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.
Abbreviations
- CCD:
-
CAROTENOID CLEAVAGE DIOXYGENASE
- CLA:
-
Carlactonoic acid
- JA:
-
Jasmonic acid
- MAX1:
-
MORE AXILLARY GROWTH 1
- MeCLA:
-
Methyl carlactonoate
- MT:
-
Methyltransferase
- SA:
-
Salicylic acid
- SAH:
-
S-Adenosyl-l-homocysteine
- SAM:
-
S-Adenosyl-l-methionine
- SL:
-
Strigolactone
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Acknowledgements
This work was supported, in part, by JST/JICA SATREPS (JPMJSA1607 to YS), JST ACT-X (JPMJAX20BM to TW), and JSPS KAKENHI (20K15459 to TW).
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425_2021_3738_MOESM1_ESM.png
Fig. S1 SDS-PAGE of recombinant At4g36470 protein. The At4g36470 was expressed as a His-tagged protein in E. coli and purified using an affinity column. M: protein marker, EV: empty vector, C: crude protein, P: purified protein (PNG 1112 kb)
425_2021_3738_MOESM2_ESM.pdf
Fig. S2 Optical resolution of CLA enantiomers. a Chiral HPLC separation of synthesized (11R)-CLA and (11S)-CLA. b Circular dichroism (CD) spectra of CLA enantiomers. The spectra of fast-moving and slow-moving isomers are drawn in red and blue, respectively (PDF 75 kb)
425_2021_3738_MOESM3_ESM.png
Fig. S3 Substrate preferences of At4g36470 were evaluated based on the SAH production, the common product for all SAM-dependent methyltransfer reactions. Error bars represent means ± SE (n = 3 biologically independent replicates). An asterisk indicates a significant difference to the control without substrate (*P < 0.05 by the Student’s t test) (PNG 318 kb)
425_2021_3738_MOESM4_ESM.png
Fig. S4 Production of MeCLA by At4g36470 in N. benthamiana. The transient expression of CLA biosynthesis genes (AtD27, AtCCD7, AtCCD8, and AtMAX1) resulted in the formation of CLA, and the co-expression of these genes along with At4g36470 produced MeCLA. MRM transitions at m/z 347.2 > 97 in positive ionization mode and m/z 331.2 > 113 in negative ionization mode were applied for the detection of MeCLA and CLA, respectively (PNG 2939 kb)
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Wakabayashi, T., Yasuhara, R., Miura, K. et al. Specific methylation of (11R)-carlactonoic acid by an Arabidopsis SABATH methyltransferase. Planta 254, 88 (2021). https://doi.org/10.1007/s00425-021-03738-6
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DOI: https://doi.org/10.1007/s00425-021-03738-6