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Soybean chalcone isomerase: evolution of the fold, and the differential expression and localization of the gene family

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Main conclusion

Soybean chalcone isomerase (CHI) family contains twelve members with unique evolutionary background, expression patterns and is compartmentalized to specific subcellular locations.

The phenylpropanoid pathway produces a diverse array of plant natural products. A key branch-point enzyme, chalcone isomerase, catalyzes the reaction producing flavanones, the backbone for many downstream metabolites such as flavonoids and isoflavonoids. We have identified twelve soybean GmCHIs that fall into four subfamilies. The study of this family in soybean in the context of various CHIs and CHI-like proteins, across divisions in the plant kingdom and beyond, shows an evolutionary journey from fatty acid-binding proteins (FAPs) to sterically restricted folds that gave rise to the chalcone-to-flavanone isomerase. There are four GmCHIs with this functionality, three of which belong to a legume-specific clade known as ‘type II’ CHIs. Tissue-specific expression of eight core members of the soybean CHI family showed differential temporal and spatial expression, pointing to the potential function of GmCHI1A in seed isoflavonoid production. Promoter analysis of the GmCHIs described the minutiae of sub-organ expression patterns. Subcellular localization of the family was conducted to investigate the possibility of pathway-specific compartmentalization. Subfamilies 1, 2 and 4 localized to the nucleus and cytoplasm, with nuclear localization of CHIs raising questions about alternate function. GmCHI3 isoforms localized to the chloroplast, which, in conjunction with their position on the phylogenetic tree and expression patterns, closely associates them with the FAPs. This study provides the first comprehensive look at soybean CHIs, a family of unique evolutionary background and biochemical function, with the catalytically active members producing the backbone substrate in an important plant metabolic pathway.

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Acknowledgments

We thank Dr. Rima Menassa (Agriculture Canada, London) for eGFP pure protein, Ling Chen, Patrick Chapman and Alex Molnar (Agriculture Canada, London) for technical assistance. This research was supported by Agriculture and Agri-Food Canada’s Abase and Genomics Research and Development Initiative grants to SD.

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Authors and Affiliations

  1. Department of Biology, University of Western Ontario, London, ON, Canada

    Mehran Dastmalchi & Sangeeta Dhaubhadel

  2. Southern Crop Protection and Food Research Center, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON, N5V 4T3, Canada

    Mehran Dastmalchi & Sangeeta Dhaubhadel

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  1. Mehran Dastmalchi
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Correspondence to Sangeeta Dhaubhadel.

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Supplementary Table 1 Sequences of oligonucleotides used in the study (DOCX 21 kb)

425_2014_2200_MOESM2_ESM.tif

Supplementary Fig. S1 Co-localization of GmCHI-YFP and organelle markers fused with CFP. (a) GmCHI1A and NLS-CFP, (b) GmCHI1B1 and NLS-CFP, (c) GmCHI1B2 and NLS-CFP, (d) GmCHI2 and NLS-CFP, (e) GmCHI3A1 and PLS-CFP, (f) GmCHI3A2 and PLS-CFP, (g) GmCHI4A and NLS-CFP, and (h) GmCHI4B and NLS-CFP. YFP, yellow fluorescent protein; CFP, cyan fluorescent protein; NLS, nuclear localization signal; PLS, plastid localization signal. Scale bar indicates 40 µm (TIFF 7100 kb)

425_2014_2200_MOESM3_ESM.tif

Supplementary Fig. S2 Subcellular localization of GmCHIs in soybean hairy roots. Constructs containing translational fusion of GmCHI-YFP were transformed into A. rhizogenes strain K599, and used in the hairy root transformation of soybean cotyledons. Confocal microscopy was used to detect expression after the emergence of hairy roots. (a) GmCHI1A, (b) GmCHI1B1, (c) GmCHI1B2, (d) GmCHI2, (e) GmCHI3A1, (f) GmCHI3A2, (g) GmCHI4A, and (h) GmCHI4B. Scale bar indicates 40 µm (TIFF 5569 kb)

425_2014_2200_MOESM4_ESM.tif

Supplementary Fig. S3 Western blot analysis of GmCHI-YFP proteins. Constructs containing translational fusions of GmCHI-YFP were transiently expressed in N. benthamiana leaves. Total soluble proteins were separated by SDS-PAGE and transferred into PVDF membrane by electroblotting. Fusion proteins were detected by sequential incubation of the blot with anti-GFP antibody and anti-rabbit IgG conjugated with horseradish peroxidase, followed by chemiluminescent reaction. eGFP fused with hydrophobin (~ 37 kDa) used as a control. All GmCHI-YFP (~ 50 kDa) appear as a single band indicating no cleavage of YFP tag. Supplementary Table 1 Sequences of oligonucleotides used in the study (TIFF 998 kb)

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Dastmalchi, M., Dhaubhadel, S. Soybean chalcone isomerase: evolution of the fold, and the differential expression and localization of the gene family. Planta 241, 507–523 (2015). https://doi.org/10.1007/s00425-014-2200-5

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