Targeted editing of multiple homologues of GTR1 and GTR2 genes provides the ideal low‐seed, high‐leaf glucosinolate oilseed mustard with uncompromised defence and yield

Abstract

SummaryGlucosinolate content in the two major oilseed Brassica crops—rapeseed and mustard has been reduced to the globally accepted Canola quality level (<30 μmoles/g of seed dry weight, DW), making the protein‐rich seed meal useful as animal feed. However, the overall lower glucosinolate content in seeds as well as in the other parts of such plants renders them vulnerable to biotic challenges. We report CRISPR/Cas9‐based editing of glucosinolate transporter (GTR) family genes in mustard (Brassica juncea) to develop ideal lines with the desired low seed glucosinolate content (SGC) while maintaining high glucosinolate levels in the other plant parts for uncompromised plant defence. Use of three gRNAs provided highly efficient and precise editing of four BjuGTR1 and six BjuGTR2 homologues leading to a reduction of SGC from 146.09 μmoles/g DW to as low as 6.21 μmoles/g DW. Detailed analysis of the GTR‐edited lines showed higher accumulation and distributional changes of glucosinolates in the foliar parts. However, the changes did not affect the plant defence and yield parameters. When tested against the pathogen Sclerotinia sclerotiorum and generalist pest Spodoptera litura, the GTR‐edited lines displayed a defence response at par or better than that of the wild‐type line. The GTR‐edited lines were equivalent to the wild‐type line for various seed yield and seed quality traits. Our results demonstrate that simultaneous editing of multiple GTR1 and GTR2 homologues in mustard can provide the desired low‐seed, high‐leaf glucosinolate lines with an uncompromised defence and yield.

Studying Salt-Induced Shifts in Gene Expression Patterns of Glucosinolate Transporters and Glucosinolate Accumulation in Two Contrasting Brassica Species

“…Glucosinolates are defence compounds and their synthesis takes place in different parts of the plant and are stored in the seed [60]. The transporters involved in the glucosinolate loading into the seed are under intensive research as they offer a means to selectively reduce glucosinolate content in the seed, without compromising their biosynthesis [39,42,43]. Reduction of glucosinolates is often a requirement for developing seed cakes for animal consumption [43,61].…”

“…Glucosinolates have also been reported to be involved in defence mechanisms against abiotic stresses by activating signalling pathways like jasmonic acid (JA) and salicylic acid (SA) pathways [38], inferring the role of these secondary metabolites in plant defence mechanisms. Disruption of glucosinolates in Brassica plants made them susceptible to disease and insect attack [39].…”

Multi-omic analyses unveil contrasting composition and spatial distribution of specialized metabolites in seeds ofCamelina sativaand other Brassicaceae

“…Hence, they have been a target of domestication for some Brassicaceae (e.g. Brassica and Camelina species) for which many efforts are still being done to modulate the content or nature of GSLs in seeds (Nour-Eldin et al, 2017;Hölzl et al, 2023;Mann et al, 2023). Nevertheless, GSLs are antioxidants accumulated in response to abiotic stresses (Rao et al, 2021;Mácová et al, 2022) and are essential for plant resistance to pathogens, so much so that their systematic elimination could prove counterproductive.…”

The C-terminal regulatory domain of IPMS enzyme maintains leucine homeostasis by bypassing a hidden negative feedback loop in plants

“…The vectors for CRISPR/Cas9-mediated mutagenesis at the C-ter region of Bju.IPMS1 homologs ( A1 and A2 ) were developed in the basic pZP200debar::SpCas9 binary vector available in the laboratory (Mann et al, 2023). The designing of 20 nt sgRNAs-1 and 2, each targeting the tenth exon of Bju.IPMS1-A1 and - A2 homologs and the development of AtU6-26 promoter:gRNA:scaffold expression cassette was performed as previously described (Mann et al, 2023). The gRNA1 and gRNA2 expression cassettes were cloned tandemly between SpeI/PacI and AscI/MfeI, respectively into the binary vector to develop the final pPZP200debar::SpCas9:gRNA1/2 gene editing construct.…”