Discoveries in model plants grown under optimal conditions can provide important directions for crop improvement. However, it is important to verify whether results can be translated to crop plants grown in the field. In this study, we sought to study the role of MYB28 in the regulation of aliphatic glucosinolate (A-GSL) biosynthesis and associated sulfur metabolism in field-grown Brassica oleracea with the use of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 gene-editing technology. We describe the first myb28 knockout mutant in B. oleracea, and the first CRISPR field trial in the United Kingdom approved and regulated by the UK Department for Environment, Food & Rural Affairs after the reclassification of gene-edited crops as genetically modified organisms by the European Court of Justice on July 25, 2018. We report that knocking out myb28 results in downregulation of A-GSL biosynthesis genes and reduction in accumulation of the methionine-derived glucosinolate, glucoraphanin, in leaves and florets of field-grown myb28 mutant broccoli plants, whereas accumulation of sulfate, S-methyl cysteine sulfoxide, and indole glucosinolate in leaf and floret tissues remained unchanged. These results demonstrate the potential of gene-editing approaches to translate discoveries in fundamental biological processes for improved crop performance.
Get full access to this article
View all access options for this article.
References
1.
HalkierBA, GershenzonJ. Biology and biochemistry of glucosinolates. Annu Rev Plant Biol. 2006; 57:303–333. DOI: 10.1146/annurev.arplant.57.032905.105228.
JugeN, MithenR, TrakaM. Molecular basis for chemoprevention by sulforaphane: a comprehensive review. Cell Mol Life Sci. 2007; 64;1105–1127. DOI: 10.1007/s00018-007-6484-5.
4.
EdmandsWM, BeckonertOP, StellaC, et al.Identification of human urinary biomarkers of cruciferous vegetable consumption by metabonomic profiling. J Proteome Res. 2011; 10:4513–4521. DOI: 10.1021/pr200326k.
5.
TrakaMH, SahaS, HusebyS, et al.Genetic regulation of glucoraphanin accumulation in Beneforte (R) broccoli. New Phytol. 2013; 198:1085–1095. DOI: 10.1111/nph.12232.
6.
KoprivaS. Regulation of sulfate assimilation in Arabidopsis and beyond. Ann Bot. 2006; 97:479–495. DOI: 10.1093/aob/mcl006.
7.
GigolashviliT, YatusevichR, BergerB, et al.The R2R3-MYB transcription factor HAG1/MYB28 is a regulator of methionine-derived glucosinolate biosynthesis in Arabidopsis thaliana. Plant J. 2007; 51:247–261. DOI: 10.1111/j.1365-313X.2007.03133.x.
8.
HiraiMY, SugiyamaK, SawadaY, et al.Omics-based identification of Arabidopsis Myb transcription factors regulating aliphatic glucosinolate biosynthesis. Proc Natl Acad Sci USA. 2007; 104:6478–6483. DOI: 10.1073/pnas.0611629104.
9.
SønderbyIE, HansenBG, BjarnholtN, et al.A systems biology approach identifies a R2R3 MYB gene subfamily with distinct and overlapping functions in regulation of aliphatic glucosinolates. PLoS ONE. 2007; 2:e1322. DOI: 10.1371/journal.pone.0001322.
10.
FrerigmannH, GigolashviliT. Update on the role of R2R3-MYBs in the regulation of glucosinolates upon sulfur deficiency. Front Plant Sci. 2014; 5:626. DOI: 10.3389/fpls.2014.00626.
11.
YatusevichR, MugfordSG, MatthewmanC, et al.Genes of primary sulfate assimilation are part of the glucosinolate biosynthetic network in Arabidopsis thaliana. Plant J. 2010; 62:1–11. DOI: 10.1111/j.1365-313X.2009.04118.x.
12.
LiF, ChenB, XuK, et al.Genome-wide association study dissects the genetic architecture of seed weight and seed quality in rapeseed (Brassica napus L.). DNA Res. 2014;21:355–367. DOI: 10.1093/dnares/dsu002.
13.
AugustineR, MajeeM, GershenzonJ, et al.Four genes encoding MYB28, a major transcriptional regulator of the aliphatic glucosinolate pathway, are differentially expressed in the allopolyploid Brassica juncea. J Exp Bot. 2013; 64:4907–4921. DOI: 10.1093/jxb/ert280.
14.
KimYB. Li X, Kim S-J, et al. MYB transcription factors regulate glucosinolate biosynthesis in different organs of Chinese cabbage (Brassica rapa ssp. pekinensis). Molecules., 2013; 18:8682–8695. DOI: 10.3390/molecules18078682.
15.
SeoM-S, JinM, ChunJ-H, et al.Functional analysis of three BrMYB28 transcription factors controlling the biosynthesis of glucosinolates in Brassica rapa. Plant Mol Biol. 2016; 90:503–516. DOI: 10.1007/s11103-016-0437-z.
16.
YiG-E, RobinAHK, YangK, et al.Identification and expression analysis of glucosinolate biosynthetic genes and estimation of glucosinolate contents in edible organs of Brassica oleracea subspecies. Molecules. 2015; 20:13089–13111. DOI: 10.3390/molecules200713089.
17.
YinL, ChenH, CaoB, et al.Molecular characterization of MYB28 involved in aliphatic glucosinolate biosynthesis in Chinese Kale (Brassica oleracea var. alboglabra Bailey). Front Plant Sci., 2017; 8:1083. DOI: 10.3389/fpls.2017.01083.
18.
ChengF, WuJ, WangX. Genome triplication drove the diversification of Brassica plants. Hort Res. 2014; 1:14024. DOI: 10.1038/hortres.2014.24.
19.
KittipolV, HeZ, WangL, et al.Genetic architecture of glucosinolate variation in Brassica napus. J Plant Physiol. 2019; 240:152988. DOI: 10.1016/j.jplph.2019.06.001.
20.
HundlebyPA, HarwoodWA. Impacts of the EU GMO regulatory framework for plant genome editing. Food Energy Secur. 2019; 8:e00161. DOI: 10.1002/fes3.161.
21.
Faure J-D NapierJA. Europe's first and last field trial of gene-edited plants?. eLife. 2018; 7:e42379. DOI: 10.7554/eLife.42379.
22.
CallawayE. EU law deals blow to CRISPR crops. Nature. 2018; 560:16.
23.
BohuonE, KeithD, ParkinI, et al.Alignment of the conserved C genomes of Brassica oleracea and Brassica napus. Theor Appl Genet. 1996; 93: 833–839. DOI: 10.1007/BF00224083.
24.
BolserD, StainesDM, PerryE, et al.Ensembl Plants: integrating tools for visualizing, mining, and analyzing plant genomic data. In: Plant Genomics Databases: Methods and Protocols. (van Dijk Aalt DJ; ed).. New York, NY: Springer. 2016; pp. 115–140. DOI: 10.1007/978-1-4939-6658-5_1.
25.
SieversF, WilmA, DineenD, et al.Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol. 2011; 7:539. DOI: 10.1038/msb.2011.75.
26.
HundlebyP, IrwinJ, WangK. Brassica oleracea and B. napus. Methods Mol Biol. 2015; 1223:287–297.
27.
LawrensonT, HundlebyP, HarwoodW. Creating targeted gene knockouts in Brassica oleracea using CRISPR/Cas9. Methods Mol Biol. 2019; 1917:155–170. DOI: 10.1007/978-1-4939-8991-1_12.
28.
LawrensonT, ShorinolaO, StaceyN, et al.Induction of targeted, heritable mutations in barley and Brassica oleracea using RNA-guided Cas9 nuclease. Genome Biol. 2015; 16:258. DOI: 10.1186/s13059-015-0826-7.
29.
EdwardsK, JohnstoneC, ThompsonC. A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucl Acids Res. 1991; 19:1349.
ParkinIA, KohC, TangH, et al.Transcriptome and methylome profiling reveals relics of genome dominance in the mesopolyploid Brassica oleracea. Genome Biol. 2014; 15:R77.
32.
RobinsonMD, McCarthyDJ, SmythGK. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 2010; 26:139–140. DOI: 10.1093/bioinformatics/btp616.
33.
AlexaA, RahnenführerJ. Gene set enrichment analysis with topGO. Bioconductor Improv. 2009; 27:1–26.
SahaS, HollandsW, TeucherB, et al.Isothiocyanate concentrations and interconversion of sulforaphane to erucin in human subjects after consumption of commercial frozen broccoli compared to fresh broccoli. Mol Nutr Food Res. 2012; 56:1906–1916. DOI: 10.1002/mnfr.201200225.
36.
KoprivovaA, NorthKA, KoprivaS. Complex signaling network in regulation of adenosine 5′-phosphosulfate reductase by salt stress in Arabidopsis roots. Plant Physiol. 2008; 146:1408–1420. DOI: 10.1104/pp.107.113175.
37.
KubecR, DadákováE. Chromatographic methods for determination of S-substituted cysteine derivatives—A comparative study. J Chromatography A. 2009; 1216:6957–6963. DOI: 10.1016/j.chroma.2009.08.032.
38.
StrackeR, WerberM, WeisshaarB. The R2R3-MYB gene family in Arabidopsis thaliana. Curr Opin Plant Biol. 2001; 4:447–456. DOI: 10.1016/s1369-5266(00)00199-0.
39.
SønderbyIE, Geu-FloresF, HalkierBA. Biosynthesis of glucosinolates-gene discovery and beyond. Trends Plant Sci. 2010; 15:283–290. DOI: 10.1016/j.tplants.2010.02.005.
40.
JiaL, CleggMT, JiangT. Evolutionary dynamics of the DNA-binding domains in putative R2R3-MYB genes identified from rice subspecies indica and japonica genomes. Plant Physiol. 2004; 134:575–585. DOI: 10.1104/pp.103.027201.
41.
DubosC, StrackeR, GrotewoldE, et al.MYB transcription factors in Arabidopsis. Trends Plant Sci. 2010; 15:573–581. DOI: 10.1016/j.tplants.2010.06.005.
42.
HofbergerJA, LyonsE, EdgerPP, et al.Whole genome and tandem duplicate retention facilitated glucosinolate pathway diversification in the mustard family. Genome Biol Evol. 2013; 5:2155–2173. DOI: 10.1093/gbe/evt162.
43.
WuS, LeiJ, ChenG, et al.De novo transcriptome assembly of Chinese kale and global expression analysis of genes involved in glucosinolate metabolism in multiple tissues. Front Plant Sci. 2017; 8:92. DOI: 10.3389/fpls.2017.00092.
44.
BraatzJ, HarloffH-J, MascherM, et al.CRISPR-Cas9 targeted mutagenesis leads to simultaneous modification of different homoeologous gene copies in polyploid oilseed rape (Brassica napus). Plant Physiol. 2017; 174:935–942. DOI: 10.1104/pp.17.00426.
45.
OkuzakiA, OgawaT, KoizukaC, et al.CRISPR/Cas9-mediated genome editing of the fatty acid desaturase 2 gene in Brassica napus. Plant Physiol Biochem. 2018; 131:63–69. DOI: 10.1016/j.plaphy.2018.04.025.
46.
SunQ, LinL, LiuD, et al.CRISPR/Cas9-mediated multiplex genome editing of the BnWRKY11 and BnWRKY70 Genes in Brassica napus L. Int J Mol Sci. 2018; 19:2716. DOI: 10.3390/ijms19092716.
47.
YangH, WuJ-J, TangT, et al.CRISPR/Cas9-mediated genome editing efficiently creates specific mutations at multiple loci using one sgRNA in Brassica napus. Sci Rep. 2017; 7:7489. DOI: 10.1038/s41598-017-07871-9.
48.
YangY, ZhuK, LiH, et al.Precise editing of CLAVATA genes in Brassica napus L. regulates multilocular silique development. Plant Biotech J., 2018; 16:1322–1335. DOI: 10.1111/pbi.12872.
49.
KirchnerTW, NiehausM, DebenerT, et al.Efficient generation of mutations mediated by CRISPR/Cas9 in the hairy root transformation system of Brassica carinata. PLoS ONE. 2017; 12:e0185429. DOI: 10.1371/journal.pone.0185429.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
