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Reduction of antinutritional glucosinolates in Brassica oilseeds by mutation of genes encoding transporters

Nature Biotechnology volume 35, pages 377–382 (2017)Cite this article

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Abstract

The nutritional value of Brassica seed meals is reduced by the presence of glucosinolates, which are toxic compounds involved in plant defense1. Mutation of the genes encoding two glucosinolate transporters (GTRs) eliminated glucosinolates from Arabidopsis thaliana seeds2, but translation of loss-of-function phenotypes into Brassica crops is challenging because Brassica is polyploid. We mutated one of seven and four of 12 GTR orthologs and reduced glucosinolate levels in seeds by 60–70% in two different Brassica species (Brassica rapa and Brassica juncea). Reduction in seed glucosinolates was stably inherited over multiple generations and maintained in field trials of two mutant populations at three locations. Successful translation of the gtr loss-of-function phenotype from model plant to two Brassica crops suggests that our transport engineering approach could be broadly applied to reduce seed glucosinolate content in other oilseed crops, such as Camelina sativa or Crambe abyssinica.

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Figure 1: Characterization of B. rapa GTR orthologs.
Figure 2: Transcriptional profiling of B. juncea GTR2 orthologs in different tissues and developmental stages using RNA sequencing (RNA-seq) analyses.
Figure 3: Identification and field characterization of stacked B. juncea gtr2 mutants.

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Acknowledgements

We thank N. MacAulay and technicians C.G. Iversen and C.C.G. Andersen (Panum Institute, Copenhagen University) for providing Xenopus oocytes, M. Burow (DynaMo Center, University of Copenhagen) for providing p-hydroxybenzyl glucosinolate and B. Haesendonckx for field trial support at Bayer. TILLING of B. rapa was performed by RevGenUK. Support for this work was provided by Danish Council for Independent Research FTP grants 09-065827/274-08-0354 (H.H.N.-E.) and 10-082395 (B.A.H.) and Danish National Research Foundation grant DNRF99 (B.A.H. and H.H.N.-E.). Novo Nordisk supported S.R.M. with a Novo Scholarship.

Author information

Author notes

  1. Hussam Hassan Nour-Eldin and Svend Roesen Madsen: These authors contributed equally to this work.

  2. Peter Denolf and Barbara Ann Halkier: These authors jointly directed this work.

Authors and Affiliations

  1. Department of Plant and Environmental Sciences, DynaMo Center, Faculty of Science, University of Copenhagen, Frederiksberg, Denmark

    Hussam Hassan Nour-Eldin, Svend Roesen Madsen, Morten Egevang Jørgensen, Jonathan Sonne Andersen & Barbara Ann Halkier

  2. Department of Plant and Environmental Sciences, Copenhagen Plant Science Center, Faculty of Science, University of Copenhagen, Frederiksberg, Denmark

    Hussam Hassan Nour-Eldin, Svend Roesen Madsen, Morten Egevang Jørgensen, Jonathan Sonne Andersen & Barbara Ann Halkier

  3. Bayer CropScience NV, Innovation Center, Zwijnaarde, Belgium

    Steven Engelen, David Seynnaeve, Thalia Verhoye & Peter Denolf

  4. Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Frederiksberg, Denmark

    Carl Erik Olsen

  5. Bayer CropScience Inc., Seed Quality Lab, Saskatoon, Saskatchewan, Canada

    Rudy Fulawka

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Contributions

S.R.M. performed most of the experiments on B. rapa and statistical analyses; M.E.J. performed TEVC measurements and phylogenetic analysis, analyzed data and made figure graphics; C.E.O. performed LC-MS analyses; J.S.A. performed RT–PCR experiments on B. rapa; H.H.N.-E. supervised the B. rapa experiments, which were designed by H.H.N.-E., S.R.M. and B.A.H.; P.D. supervised the B. juncea experiments, which were designed by P.D. and S.E.; P.D., S.E., T.V. and D.S. performed and analyzed the B. juncea experiments and analyzed field trial data; R.F. performed lipid analysis on B. juncea and B. rapa seeds; D.S. performed uptake analyses on B. juncea and B. napus GTRs under supervision by H.H.N.-E.; H.H.N.-E., S.R.M., M.E.J., S.E., P.D. and B.A.H. wrote the paper from a draft written by S.R.M. and H.H.N.-E.

Corresponding authors

Correspondence to Peter Denolf or Barbara Ann Halkier.

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Competing interests

Transport engineering of glucosinolates is the subject of a patent (WO2012004013 on January 12, 2012) published by P.D., C.O., B.A.H., H.H.N.-E., T.G.A., S.R.M. and M.E.J.

Integrated supplementary information

Supplementary Figure 1 Characterization of B. rapa GTRs.

(a) Expression analysis by RT-PCR of BrGTR2A1, BrGTR2A2, BrGTR2A3 and BrGTR2A4 in wildtype B. rapa tissues. Reference gene UBC21. PCR reactions are representative of two independent repetitions on the same cDNA preparation (b) I-V (Current-Voltage) curve of 4MTB-dependent currents for a BrGTR2A2-expressing oocyte before (filled circle), during (empty circle) and after (filled triangle) exposure to 100μM 4MTB pH5. Data shown representative of 5 oocytes from one frog. Results representative of two independent experiments (i.e. 5 additional oocytes from another frog). (c) Determination of Km value for BrGTR2A2 by two-electrode voltage-clamp measurements in Xenopus oocytes. Normalized 4MTB-dependent currents measured at a membrane potential of -60mV and pH 5 were plotted against increasing 4MTB concentrations. Each oocyte dataset was normalized to currents elicited at 100 μM 4MTB at -60mV. The saturation curve was fitted with a Michaelis-Menten equation. Insert: voltage-dependency of Km constant. Error bars are s.e.; n=6 oocytes. Results representative of two independent experiments.

Supplementary Figure 2 Investigations of B. rapa and B. juncea gtr mutant plant lines

HPLC analyses of glucosinolate concentration in seeds from single representative B. rapa lines of native wildtype, Brgtr2A2stop and BrGTR2A2WT in the M7 generation. n= 5 plants for native wildtype and Brgtr2A2stop, n=2 plants for BrGTR2A2WT (12 seeds collected from each plant). Different letters indicate statistically significant differences (One-way ANOVA, P < 0.05). Results are representative of two independent experiments. (b) Glucosinolate concentration in seeds from field-grown B. juncea (mutant population 1 (see also Supplementary Table 4)). Each value represents the average of four replicates in three locations for each genotype. Statistical difference (p< 0.05) was calculated using a linear mixed model between segregating wildtype and mutants (*). (c) Average weight of M7 seeds from single representative lines of native wildtype, Brgtr2A2stop and BrGTR2A2WT. n= 5 plants for native wildtype and Br gtr2A2stop, n=2 plants for BrGTR2A2WT (12 seeds collected from each plant). No statistically significant differences (One-way ANOVA, P>0.05). (d) Analysis of fresh weight of tissues from single representative M5 B. rapa lines of native wildtype, BrGTR2A2WT and Br gtr2A2stop. n= tissue from 5 plants. Averages with the same letter within grouped columns (indicated by braces: Leaves, Siliques, Stem, Root) are not significantly different from each other. (One-way ANOVA, P < 0.05). Data in all subpanels are box-and-whisker representations showing the 10th, 25th, 75th and 90th percentiles. The line within boxes is the data median. Any outliers are shown as individual data points.

Supplementary Figure 3 3-Butenyl glucosinolate (3-But) uptake of B. juncea and B. napus GTR1- and GTR2-expressing Xenopus oocytes

Each B. juncea GTR (a) or B. napus GTR (b) was expressed in Xenopus oocytes and transport activity measured in the presence of saturating 3-But concentrations. Imported 3-But into oocytes for each gene is depicted relative to content in AtGTR1-expressing oocytes (n=3 (each consisting of extracts from 5 pooled oocytes). Data is represented as box-and-whisker plots showing the 10th, 25th, 75th and 90th percentiles. The line within boxes is the data median. Any outliers are shown as individual data points. Results are representative of two independent experiments.

Supplementary Figure 4 Transcriptional profiling of Brassica juncea GTR1 orthologs in different tissues at a range of developmental stages

Expression profiles of BjGTR1 orthologs in meristematic, vegetative tissues and reproductive tissues. Selected tissues of greenhouse-grown B. juncea were collected at certain intervals (days after sowing (DAS) or days after flowering (DAF)). Total mRNA was extracted and gene expression was quantified and stated as CPKM (Counts Per Kilobase per Million reads). Every experiment has been replicated 3 times; all data from these 3 replicates have been merged as 1 dataset (see methods).

Supplementary Figure 5 Expression analysis of BrGTR2 genes

Expression analysis by RT-PCR of BrGTR2A1, BrGTR2A2, BrGTR2A3 and BrGTR2A4 in wildtype and Brgtr2A2stop (KO) B. rapa tissues. Reference gene UBC21 (ubiquitin-conjugating enzyme 21). Shown is two versions of the full size gel picture from which the

cropped gel picture shown in Supplementary Figure 1a has been generated. Supplementary figure 1a shows only the bands from the WT line. Top part shows the annotated cropping of the bottom gel which shows the raw gels (note that BrGTR2A2 is depicted above BrGTR2A1 on this gel). PCRs were run on two PCR machines. The reference gene UBC21 was included on each machine.

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Nour-Eldin, H., Madsen, S., Engelen, S. et al. Reduction of antinutritional glucosinolates in Brassica oilseeds by mutation of genes encoding transporters. Nat Biotechnol 35, 377–382 (2017). https://doi.org/10.1038/nbt.3823

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