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. 2025 Jun 11;34(1):29. doi: 10.1007/s11248-025-00447-8

Mutation in soybean Lox-2 PLAT/LH2 domain through CRISPR/Cas9 reduces seed lipoxygenase activity: responsible for undesirable flavour

Ekta Patel 1, Piyali Das 1, Somak Hazra 1, Manveer Sharma 1, Gautam Chhabra 1, Balwinder Singh Gill 2, Sucheta Sharma 3, Ajinder Kaur 1, Deepak Singla 1, Jagdeep Singh Sandhu 1,✉
PMCID: PMC12158852  PMID: 40498336

Abstract

Soybean, a protein and oil rich legume is primarily used as livestock feed and to a lesser extent for human consumption due to undesirable flavour in the seeds caused by L-2 isozyme of lipoxygenase. Herein, soybean with reduced isozyme activity was developed through CRISPR/Cas9 targeted mutation in L-2 encoding Lox-2 gene. sgRNA designed from PLAT/LH2 domain in second exon of Lox-2 (Lox-2 E2) was validated by in vitro cleavage assay; inserted in CRISPR/Cas9 binary vector and used for genetic transformation of SL1074 cultivar hypocotyl segments. A total of 12 T0 putative plants were identified through PCR. Amongst these, four revealed mutation at the target sgRNA site by CEL1 assay and substitution of a base A with G six bp upstream of PAM converting lysine to glutamic acid at 119 position. T1 and T2 seeds derived from mutant T0–37 plant showed upto 25.49% reduction in isozyme activity as compared to SL1074. The base substitution was confirmed in T1 progeny; segregation analysis revealed homozygosity and heritability of mutation in T2 plants. The interaction between structural models of SL1074, mutant domains and negatively charged substrates revealed strong binding affinity of the substrates with positively charged lysine in SL1074 domain due to formation of two hydrogen bonds. On the contrary, weak binding of the substrates with negatively charged glutamic acid in mutant domain and absence of hydrogen bond explained reduction of isozyme activity in T2 seeds. The mutant soybean with reduced isozyme activity is an important source for introgressing the trait in plant breeding programs.

Supplementary Information

The online version contains supplementary material available at 10.1007/s11248-025-00447-8.

Keywords: Glycine max L., Genome editing, Base substitution, Isozyme activity, Structural modelling

Introduction

Soybean (Glycine max L.) is a protein (40%) and oil (20%) rich leguminous crop. Globally, two-third of the crop produce is used as livestock feed and one-fourth for human consumption (Agarwal et al. 2013). One of the reasons for low consumption by humans is the presence of undesirable flavour in soybean seeds that is mainly formed by lipid oxidizing enzyme, lipoxygenase (EC 1.13.11.12) (Matoba et al. 1985). Three distinct lipoxygenase isozymes are present in soybean seeds viz., lipoxygenase-1 (L-1), lipoxygenase-2 (L-2) and lipoxygenase-3 (L-3) (Matoba et al. 1985). The isozymes oxidize fatty acids and form 9-, 13-hydroperoxides, on dioxygenation these produce volatile compounds (such as, aldehydes, ketones and n-hexanal) linked with the development of undesirable flavour (Davies et al. 1987). Among the three isozymes, L-2 is mainly responsible for the production of n-hexanal in the seed (Matoba et al. 1985). The gene encoding L-2 (Lox-2) has been characterized, and targeted mutation in Lox-2 has led to generation of soybean mutants with less pronounced undesirable flavour (Wang et al. 1994; Reinprecht et al. 2011).

Mutagenesis is an integral part of crop improvement as it creates heritable genetic variation. The application of chemical, physical and biological mutagens cause spontaneous and random mutations that are difficult to use in plant breeding programs (Oladosu et al. 2016). On the contrary, CRISPR/Cas9 is an exceptionally efficient tool to mutate the target gene using sequence specific nucleases that generate double stranded breaks in DNA leading to deletions, insertions and/or substitutions in the gene (Rao et al. 2022). The tool has been successfully used in soybean for modifying flowering time by targeting GmFT2a gene (Cai et al. 2018), increasing oleic acid, lowering linoleic and α-linolenic acid content by disrupting GmFAD2 genes (Amin et al. 2019; Do et al. 2019; Li et al. 2023; Zhou et al. 2023), improving plant architecture by editing GmSPL9 and GmDWF1 genes (Bao et al. 2019; Xiang et al. 2024), generating lipoxygenase-free mutants by knocking out GmLox genes (Wang et al. 2020), inducing male sterility by editing GmAMS1 (Chen et al. 2021), reducing phytic acid content by disrupting GmIPK1 (Song et al. 2022), enhancing aroma by editing GmBADH2 (Qian et al. 2022), increasing seed size by disrupting GmEOD1 (Yu et al. 2023), enhancing resistance to powdery mildew, soybean mosaic virus and blast by targeting MLO, HC-Pro and GmTAP1 genes, respectively (Bui et al. 2023; Liu et al. 2023; Gao et al. 2024) and improving resistance against multiple stresses by editing GmARM (Luo et al. 2024). These reports demonstrated application of CRISPR/Cas9 to genetically improve the agronomic traits for which limited variation is available in the germplasm.

The present study was undertaken to develop soybean with reduced seed lipoxygenase activity by targeting PLAT/LH2 domain in second exon of Lox-2 gene in a commercial cultivar SL1074 through CRISPR/Cas9. The substitution of a base A with G six bp upstream of PAM was detected that caused conversion of lysine to glutamic acid at 119 position resulting in up to 25.93% reduction in isozyme activity in mutant T2 seeds as compared to SL1074. The interaction between structural models of SL1074, mutant domains and negatively charged substrates revealed strong binding affinity of the substrates with positively charged lysine in SL1074 domain as compared to weak binding of the substrates with negatively charged glutamic acid in mutant domain, explaining reduction of isozyme activity in T2 seeds.

Materials and methods

Plant material

The mature seeds of a high yielding and early maturing soybean cultivar SL1074 were procured from the Department of Plant Breeding and Genetics, Punjab Agricultural University, Ludhiana, Punjab, India during 2021–2022. The seeds were germinated in vitro to obtain 8-day old seedlings that were used to prepare explants from hypocotyls for agro-infection (Kantayos and Bae 2019).

In silico analysis of soybean Lox-2 gene and designing of sgRNA

The genomic sequence of Lox-2 (GenBank Accession No. GU942744) was fetched from NCBI database. The sequence was used for designing exonic region 2 (Lox-2 E2) specific PCR primers i.e. 5′-GTGTATGTATGGTCTGTTTGTAGC-3′ (forward) and 5′-GGATATTCGACCAGTTCTTCCC-3′ (reverse). The primers were applied to amplify Lox-2 E2 region from genomic DNA of SL1074 cultivar. The amplicon was Sanger sequenced (ZumZum Biochemicals, Hyderabad) and Lox-2 E2 specific sgRNAs were designed using online CRISPR-Pv2.0 tool (Liu et al. 2017). During designing, the parameters such as, length of guide sequence, per cent GC content, off-targets and on-score were considered. The list of candidate sgRNA sequences is shown in Table S1.

In vitro synthesis of sgRNA and assessment of cleavage efficiency

The sgRNA-DNA template was synthesized in vitro by annealing sgRNA oligonucleotides after adding T7 promoter sequence using GeneArt Precision gRNA Synthesis Kit (Thermofisher, USA). The reaction mixture (25.0 µl) comprised of Phusion-High-Fidelity PCR master mix (12.5 µl), Tracr fragment + T7 primer mix (1.0 µl), 10 µM of annealed oligonucleotide mix (1.0 µl) and nuclease-free water (10.5 µl). The reaction was carried out with initial denaturation at 98 °C for 10 s, followed by denaturation at 98 °C for 5 s that was repeated 32 times, annealing at 55 °C for 15 s, final extension at 72 °C for 15 s in a Veriti 96-well thermocycler (Applied Biosystems, Thermofisher Scientific, USA). The template was electrophoresed at 80 V for 1 h on ethidium bromide stained agarose gel (2%) using PowerPac™ HC (BioRad, USA). The gel was visualized under UV light and captured using gel documentation system (Avegene, USA).

In vitro synthesis of sgRNA was carried out in 20.0 µl reaction using GeneArt Precision gRNA Synthesis Kit (Thermofisher, USA). The reaction mixture comprised of template (6.0 µl), dNTP mix (8.0 µl), transcript aid enzyme mix (2.0 µl) and 5 × transcript reaction buffer (4.0 µl). The mixture was incubated at 37 °C for 3 h. Thereafter, purification of sgRNA was carried out using commercial wash buffer containing 96% ethanol, followed by centrifugation (Remi, India) at 14,000 rpm. The purified sgRNA was eluted using elution buffer and verified through electrophoresis. sgRNA and Cas9 endonuclease (New England Biolabs, USA) were mixed in 1:1 ratio to assess in vitro cleavage efficiency of Lox-2 E2 amplicon.

Vector construction

sgRNA was cloned in CRISPR/Cas9 pKSE401vector (Adgene, USA) following Golden gate cloning (Weber et al. 2011). sgRNA oligonucleotides (5′-GATTTGAATGGGACGAAAGCATGG-3′ and 5′-AAACCCATGCTTTCGTCCCATTCA-3′) were synthesised by incorporating BsaI adapters (underlined) at 5′ position of forward and reverse strands. The oligonucleotides were annealed by adding T4 polynucleotide kinase (1.0 µl), T4 DNA ligase buffer (1.0 µl) and nuclease-free water (6.0 µl). The annealing was carried out by incubating the reaction mixture at 37 °C for 60 min, followed by sequential incubation at 95 °C, 72 °C and 25 °C for 10 min on each step. The duplex oligonucleotides were ligated in BsaI digested vector. The ligation was verified through colony PCR using universal M13 forward primer 5′-GTTTTCCCAGTCACGACGTTGTA-3′ and sgRNA reverse primer following the amplification conditions as explained earlier. The plasmid DNA was isolated from PCR positive colonies and double digested using restriction enzymes. The digestion mixture (10.0 µl) contained isolated plasmid DNA (5.0 µl), SpeI (1.0 µl), XmaI (1.0 µl), cut smart buffer (1.0 µl) and nuclease-free water (2.0 µl), followed by incubation at 37 °C for 2 h. The mixture was electrophoresed and recombinant vectors were Sanger sequenced. The confirmed vector was mobilized into Agrobacterium strain EHA105 using freeze thaw method (Holsters et al. 1978). The transformed Agrobacterium was spread on Luria Bertani (LB) Agar (HiMedia, Mumbai) supplemented with kanamycin (50 mg/l) and rifampicin (50 mg/l) and incubated at 28 °C for 48 h. The recombinant Agrobacterium colonies were verified by PCR using sgRNA specific primers.

Agrobacterium-mediated transformation of soybean

A single Agrobacterium colony was used to inoculate liquid LB medium (5.0 ml) supplemented with antibiotics for preparing suspension by incubating at 28 °C till OD650nm reached at 0.8 (Yang et al. 2016). The suspension was centrifuged (Remi) at 4500 rpm for 10 min and the pellet was suspended in liquid MS medium (30.0 ml) containing sucrose (30 g/l) (pH 5.8). The transversely cut soybean hypocotyl segments were agro-infected for 30 min, followed by washing with sterile water and blot-dried on sterile filter paper (Whatman, USA). The segments were transferred to co-cultivation medium [MS supplemented with acetosyringone (100 mM) l-glutamine (100 mM), sucrose (30 g/l) and agar (8 g/l), pH 5.8] in petri plates (90 mm, Tarsons, India) and incubated under 16 h photoperiod at 27 ± 2 °C for 72 h. The co-cultivated segments were transferred to direct shoot regeneration medium for shoot emergence as described by Patel et al. (2023). The regenerated shoots with adventitious roots were hardened on water moistened cotton kept in glass jars that were covered with polythene sheets and incubated for 3 days. Subsequently, the hardened plantlets were transferred to earthen pots containing garden soil mixed with cocopeat (1:1) and maintained in transgenic glass house at 28–30 °C with watering twice a day.

Screening of putative transformed plants

The genomic DNA was isolated from tender leaves of 12-week-old putative transformed and wild type plants using CTAB method (Saghai-Maroof et al. 1984). The putative plants were identified by analyzing the presence of CaMV 35S promoter region of gene cassette in genomic DNA using promoter specific forward 5′-GGCCCATTTAAGTTGAAAACAATC-3′ and reverse 5′-GCACGACACTCTCGTCTACTC-3′ primers through PCR. The plants positive for transformation were further analyzed using CEL1 endonuclease assay.

Detection of mutation in Lox-2 E2 using CEL1 assay

Lox-2 E2 region DNA from 12-week-old transformed plants was amplified through PCR using gene specific primers. The amplicons were purified using purification kit (Macherey Nagel, Germany), and analyzed by CEL1 endonuclease assay. The assay mixture (10.0 µl) comprising of purified DNA samples (6.0 µl, 50 ng/µl), CEL1 enzyme (1.0 µl, 100 U/µl), reaction buffer (1.0 µl, 2 ×), purified water (3.0 µl) was incubated in Veriti 96 well thermocycler at 37 °C for 1 h. The mixture was cooled on ice for 5 min and subsequently resolved on 2% agarose gel to examine the DNA cleavage. The samples exhibiting cleavage were Sanger sequenced (Barcode Biosciences, Hyderabad) for the identification of mutations within sgRNA region. The order of amino acids encoded by the mutated sequences was determined using Expasy tool (Swiss Institute of Bioinformatics).

Quantitative estimation of Lox-2 isozyme activity from the soybean seeds

Lox-2 activity was quantitatively estimated according to the protocol given by Axerold et al. (1981). A total of five soybean seeds (picked randomly) from each mutant were crushed separately into fine powder that was homogenized in sodium phosphate buffer (pH 6.8) for extracting the isozyme. The reaction mixture (1760 µl) comprised of extracted isozyme (10 µl), sodium tetraboratein buffer supplemented with borax (1500 µl) and purified linolenic acid (250 µl). The change in absorbance was monitored in a UV spectrophotometer (Agilent technologies, USA) at 234 nm every 30 s till 3 min. The increase in the rate of absorbance was used for quantification of isozyme activity; one unit of activity was defined as the amount of isozyme yielding rise in absorbance (1.0/min) at 234 nm and calculated using the formula given by Deshpande et al. (2015).

Δabsorbance permin×volume of reaction×1000×supernatantε×1000×enzyme volume×tissue weight

where Δ absorbance per min: mean difference in the absorbance readings; volume of reaction: enzyme (10 µl) + buffer (1500 µl) + linolenic acid (250 µl), supernatant: volume of seed extract (850 µl); ε:27,500; enzyme volume: volume of enzyme used (10 µl), tissue weight: weight of seed extract (g). The experiment was performed in triplicates and the results were compared with the wild type SL1074 cultivar. Normality of the data was assessed using Shapiro–Wilk test and equality of variances was evaluated by F-test followed by paired t test. The statistical significance was determined using least significance difference t test at P ≤ 0.05.

Structural modelling and docking of Lox-2 PLAT/LH2 domain

The conserved nucleotide sequence of PLAT/LH2 domain (template: pdbID d1rrha2) was translated using Expasy tool. The 3D structure of the protein was predicted through Phyre2, followed by validation with Ramachandran plot. The 3D structures of substrates, linoleic acid (PubChem ID: 5280450) and arachidonic acid (PubChem ID: 444899) were fetched and converted in PDB format. The predicted structures of the domain and the substrates were energy minimized using UCSF Chimera v1.16. The mutation site-specific docking was performed by AutoDock tool v1.5.7. The substrates and domain were prepared by addition of hydrogen ions, gasteiger and Kollman charges, merging of non-polar hydrogen atoms, defining AD4 atom types, etc. The 3D grid for wild type and mutant PLAT/LH2 domains were generated with coordinates (− 11.770, 13.124 and − 6.984 Å) and (− 14.072, 12.744 and − 0.319 Å), respectively at the target site with box size (52, 44, 58) by AutoGrid. The docking was performed using Lamarckian Genetic Algorithm with 20 runs, maintaining the domain in rigid state, but allowing substrate to remain flexible. The binding affinity of substrate-domain complex was screened in AutoDock, and hydrogen bond interaction was visualized on PDBSum server.

Statistical analysis

Lox-2 enzyme activity was calculated according to Deshpande et al. (2015); the data are represented as mean ± SD of three replicates, and significance was tested using two-tailed paired t test. The calculated p value < 0.05 was regarded as significant. The segregation data was analyzed for statistical significance by Chi-square test; calculated value more than table value was regarded as significant at 5% level of significance.

Results

sgRNA designing for inducing targeted mutation

sgRNA sequences were designed from Lox-2 E2 as a target for Cas9 endonuclease (Fig. 1a). The sequence 5′-TGAATGGGACGAAAGCATGG-3′ having PAM motif (designated as Lox-2 E2 sg_2 and herein after referred to as sgRNA) displaying 50% GC content, 30 off-targets, and 0.30 on-score was selected for inducing desired mutation (Table S1). The prediction of Lox-2 E2 sg_2 secondary structure revealed 2–3 loops in the complex with tracrRNA indicating enhanced stability, performance, and ability of sgRNA to assist cleavage by Cas9 (Fig. S1). The efficacy of Cas9 for cleaving target site was evaluated using in vitro assay.

Fig. 1.

Fig. 1

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CRISPR/Cas9 vector. a Structure of Lox-2 coding and non-coding regions highlighting exon 2 (E2) selected for designing sgRNA, and b view of CRISPR/Cas9 vector carrying Lox-2 sgRNA

In vitro assay of Lox-2 E2 with Cas9 endonuclease for cleavage efficiency

The specificity of sgRNA in Lox-2 E2 with Cas9 was verified by in vitro cleavage assay. The incubation of Lox-2 E2 amplicon (686 bp) with Cas9 led to generation of two fragments. The fragments were of the anticipated size i.e., 530 bp and 156 bp that occurred due to cleavage within the sgRNA pointing towards specific action of Cas9 on Lox-2 E2 sgRNA. The results demonstrated the ability of Cas9 to cause double stranded breaks at the Lox-2 E2 sgRNA target site (Fig. 2).

Fig. 2.

Fig. 2

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In vitro assay of Lox-2 E2 with Cas9 endonuclease for cleavage efficiency. Lane 1 represents non-cleaved Lox-2 E2 amplicon (686 bp) without incubation with Cas9; Lane 2 depicts cleaved amplicon upon incubation with Cas9 displaying two fragments (530 bp and 156 bp); L represents BenchTop 100 bp ladder (SMOBIO, Cat No. DM2100)

Genetic transformation and PCR analysis for identification of T0 putative plants

CRISPR/Cas9 binary vector pKSE401 harbouring sgRNA was constructed. The sgRNA was placed under the control of Arabidopsis thaliana U6 promoter and Cas9 was driven by 2 × CaMV 35S promoter (Fig. 1b). The vector was mobilized into Agrobacterium strain EH105 that was used for infecting hypocotyl segments of soybean cultivar SL1074 (Fig. 3). A total of 1807 agro-infected segments were transferred for shoot regeneration. The direct shoot bud induction was observed from the acropetal ends in 938 segments and the shoot formation was recorded in 509 segments (Table S2) the toot formation was observed in 226 shoots. Eventually, 43 putative plantlets were transferred to soil and analyzed for the presence of CaMV 35S promoter region in genomic DNA. The results revealed amplification of 359 bp fragment corresponding to the promoter in 12 T0 plants designated as T0–5, T0–8, T0–9, T0–10, T0–20, T0–25, T0–27, T0–28, T0–30, T0–37, T0–40 and T0–43 (Fig. S2). The plants displayed normal growth and development.

Fig. 3.

Fig. 3

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Agrobacterium-mediated genetic transformation of soybean. a Agro-infected hypocotyl segments, b shoot bud induction, c shoot formation, d shoot proliferation, e root induction, and f transfer to soil

CEL1 assay on PCR positive T0 plants for detection of mutation

Lox-2 E2 amplicons from 12 T0 plants were analyzed for detection of mutation in sgRNA using CEL1 endonuclease assay. The incubation of amplicons (686 bp) with CEL1 resulted in cleavage and formation of 530 bp and 156 bp sized fragments in T0–10, T0–25, T0–28 and T0–37 plantlets indicating specific action of the endonuclease on Lox-2 E2 sgRNA (Fig. 4). The amplicons from remaining plants did not show digestion on incubation with CEL1. Thus, the results exhibited occurrence of mutation at the target sgRNA site in four T0 plants.

Fig. 4.

Fig. 4

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CEL1 assay on PCR positive T0 plants. Lanes T0–5 to T0–43 represent amplicons from PCR positive plants incubated with CEL1; arrows indicate cleavage and formation of 530 bp and 156 bp sized fragments; L represents BenchTop 100 bp ladder (SMOBIO, Cat No. DM2100); NE depicts non-edited plant incubated with CEL1

Sequencing of T0 plants for identification of mutations

Lox-2 E2 amplicons from 12 T0 plants analyzed for CEL1 assay were sequenced using E2 specific primers. The chromatograms of four plants where CEL1 displayed action on sgRNA indicated substitution of a base i.e., A with G 6 bp upstream of PAM sequence (Fig. 5a, b). On the contrary, the chromatograms of eight plants (T0–5, T0–8, T0–9, T0–20, T0–27, T0–30, T0–40 and T0–43) where CEL1 failed to cleave the amplicons did not exhibit any base change within the sgRNA (Fig. S3). The determination of amino acid order encoded by the mutated sequences revealed the conversion of lysine (K)–glutamic acid (E) due to substitution of base A with G in the sgRNA (Fig. 5c). The mutant T0 plants were grown to maturity and T1 seeds were used for (1) quantification of Lox-2 isozyme activity and (2) raising of T1 progeny for segregation analysis and sequencing.

Fig. 5.

Fig. 5

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Sequencing of T0 plants. a Chromatograms showing base substitution in sgRNA (blue box); the numbers T0–8, T0–25, T0–28 and T0–37 represent T0 plants; the sequence in red box depicts PAM, b mutation in Lox-2 E2 target site and c change of amino acid corresponding to the mutated base. (Color figure online)

Evaluation of T1 seeds for quantification of Lox-2 isozyme activity

T1 seeds collected from each self-pollinated mutant T0 plant were quantitatively evaluated for Lox-2 isozyme activity. The mean isozyme activity in five randomly picked T1 seeds from every T0 plant T0–10, T0–25, T0–28 and T0–37 was 351.48 ± 29.54, 358.70 ± 24.37, 374.65 ± 38.82 and 329.60 ± 28.79 units/g, respectively as compared to 436.36 ± 19.59 units/g in seeds of wild type plant SL1074 (Tables S3, S4). The significantly lower isozyme activity in T1 seeds pointed towards editing of Lox-2, however the isozyme activity could be skewed due to analysis of randomly selected non-edited seeds. The remaining T1 seeds were sown to raise T1 progeny plants that were evaluated for segregation and sequencing (Table S5).

Segregation analysis of T1 progeny plants for inheritance of mutation

The inheritance pattern of mutation was determined in T1 progeny plants through segregation analysis by PCR. The progeny plants derived from four T0 mutants (T0–10, T0–25, T0–28 and T0–37) were designated as T1–10–1, T1–10–2 and T1–10–3; T1–25–1, T1–25–2, T1–25–3, T1–25–4 and T1–25–5; T1–28–1, T1–28–2, T1–28–3 and T1–28–4; T1–37–1, T1–37–2, T1–37–3, T1–37–4 and T1–37–5 (Table S5). The presence of CaMV 35S promoter sequence in genomic DNA was detected in 13 progeny plants (T1–10–1, T1–10–3; T1–25–2, T1–25–3, T1–25–5; T1–28–1, T1–28–3, T1–28–4; T1–37–1, T1–37–2, T1–37–3, T1–37–4 and T1–37–5) (Fig. S4) revealing segregation in the ratio of 2:1, 3:2, 3:1 and 5:0 in T1–10, T1–25, T1–28 and T1–37 plants, respectively (Table S5). The segregation of progenies in the expected Mendelian ratio (3:1) revealed that the mutations were heritable. The mutant T0–37 plant along with its progeny was homozygous for CaMV 35S promoter (Table S5). Further, the inheritance of mutated bases in 13 progeny plants was tracked through sequencing; the chromatograms exhibited substitution of A with G in 12 T1 plants i.e. T1–10–3, T1–25–2, T1–25–3, T1–25–5, T1–28–1, T1–28–3, T1–28–4, T1–37–1, T1–37–2, T1–37–3, T1–37–4 and T1–37–5 confirming (1) inheritance of mutation, and (2) T0–37 plant and its progeny to be homozygous for base substitution (Fig. S5). T1–10–1 plant did not show substitution and was comparable to non-edited plant SL1074. T2 seeds collected from base substituted mutant T1 plants i.e. T1–37–1, T1–37–2, T1–37–3, T1–37–4 and T1–37–5 were quantified for Lox-2 isozyme activity, and the T2 seedlings were evaluated for segregation of mutation.

Assessment of T2 seeds for quantification of Lox-2 isozyme activity

T2 seeds collected individually from self-pollinated mutant T1–37 plants were quantified for Lox-2 isozyme activity. The mean isozyme activity in five randomly taken T2 seeds from each T1 plant T1–37–1, T1–37–2, T1–37–3, T1–37–4 and T1–37–5 was 325.74 ± 29.93, 329.29 ± 28.72, 329.91 ± 26.00, 326.06 ± 30.05 and 327.51 ± 28.70 units/g, respectively in comparison to 439.81 ± 37.90 units/g in wild type plant SL1074 seeds (Fig. 6; Tables S6, S7). The isozyme activity in T2 seeds was significantly less than SL1074 (p < 0.05) with up to 25.93% reduction. The remaining seeds were used to raise T2 progeny plants for analyzing segregation of mutation.

Fig. 6.

Fig. 6

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Quantification of Lox-2 isozyme activity in T2 seeds. The bars represent mean standard error

Segregation analysis of T2 progeny plants for determining inheritance pattern of mutation

The inheritance pattern of mutation was determined in T2 progeny plants, namely T2–37–P1, T2–37–P2, T2–37–P3, T2–37–P4 and T2–37–P5 derived from T1–37 plant exhibiting reduction in Lox-2 activity. The genomic DNA of 128 progeny plants was analyzed for the presence of CaMV 35S promoter sequence through PCR. The results revealed amplification of promoter region in all the progeny plants confirming homozygous nature of the progeny (Fig. S6; Table S8).

Structural modelling and docking of PLAT/LH2 domain for analyzing binding affinity

The structural models of wild type and mutant domains were analyzed for their binding affinities with two substrates (linoleic acid and arachidonic acid). As a preliminary step, Lox-2 PLAT/LH2 conserved domain model was developed that showed 89% identity, 100% coverage, 2.0 Å resolution for 177 residues with wild type and the mutant domains. The structural validation revealed 83.5% residues in most favoured region, 13.7% in additional allowed region, 2.9% in generously allowed region and no residue in disallowed region (Fig. S7). Docking of wild type and mutant PLAT/LH2 domains with linoleic acid exhibited binding affinities of − 3.53 and − 4.71 kcal/mol, respectively (Fig. 7a, b). The interaction of lysine (K119) in wild type domain with linoleic acid was through single hydrogen bond (2.63 Å) (Fig. 7c), whereas, in the mutant domain (carrying glutamic acid E119 in place of lysine), no hydrogen bond was detected between glutamic acid and the substrate (Fig. 7d). Likewise, docking of wild type and mutant domains with arachidonic acid displayed binding affinities of − 3.46 and − 4.11 kcal/mol, respectively (Fig. 7e, f). Additionally, two hydrogen bonds (2.63 Å and 2.93 Å) were identified between lysine in wild type domain and arachidonic acid (Fig. 7g), in contrast, glutamic acid in the mutant domain and arachidonic acid did not display hydrogen bonding (Fig. 7h). The results demonstrated weak binding affinity of mutant PLAT/LH2 domain with both the substrates as compared to strong affinity of substrates with wild type domain.

Fig. 7.

Fig. 7

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Docking and interaction of PLAT/LH2 domains with substrates at the target mutation site. Docking of a wild type protein domain with linoleic acid, b MUTANT domain with linoleic acid. Interaction of c lysine in wild type protein domain showing hydrogen bonding (green arrow) with linoleic acid, d glutamic acid in mutant domain with linoleic acid. Docking of e wild type protein domain with arachidonic acid, f mutant domain with arachidonic acid. Interaction of g lysine in wild type protein domain displaying two hydrogen bonds (green arrows) with arachidonic acid, h glutamic acid in mutant domain with arachidonic acid. (Color figure online)

Discussion

This study highlights the development of soybean mutants with reduced lipoxygenase enzyme activity in the seeds. The isozyme L-2 of lipoxygenase is known to be responsible for the development of undesirable flavour in soybean seeds and this characteristic limits the consumption of soy-products (Lozano et al. 2007). Herein, we report targeted mutation in the gene encoding L-2 (Lox-2) through CRISPR/Cas9 editing that led to reduction of isozyme activity in T2 seeds of a soybean cultivar SL1074, commercially grown on a large area in North and North-Eastern India.

Soybean Lox-2 protein contains two catalytic domains, i.e. polycystin-1 lipoxygenase α-toxin domain or lipoxygenase homology (PLAT/LH2) domain at the N-terminus and a highly conserved lipoxygenase domain at the C-terminus (Tolley et al. 2018). The mutation in these domains can result in alteration of Lox-2 activity (Mandal 2013). In this study, targeting of PLAT/LH2 domain displayed significant reduction (25.49%) in Lox-2 isozyme activity. The domain regulates lipoxygenase catalytic activity through membrane binding and by varying substrate specificity (Ivanov et al. 2010; Joshi et al. 2013). The loss of lipoxygenase activity following mutation in PLAT/LH2 domain of soybean Lox-2 gene has also been reported by Wang et al. (2020).

The sequencing of Lox-2 (second exon) from mutated T2 seeds demonstrated substitution of a base i.e. A with G 6 bp upstream of PAM motif in four independent lines. The occurrence of identical mutation is likely due to gRNA specificity; this hypothesis draws support from Bertier et al. (2018) where gRNA specificity has been attributed to cause alike mutations in multiple T2 plants across independent lines. The substitution of A with G in resulted in conversion of lysine (K) to glutamic acid (E) at 119 position, pointing towards its probable role in regulating Lox-2 isozyme activity. Bateman and Sandford (1999) reported the presence of conserved lysine residues at K100, K114, and K126 in PLAT/LH2 domains of all lipoxygenase isoforms in soybean and their involvement in associating isozymes with membrane (negatively charged) and substrates (lipoprotein-sequestered). In present study, weak electrostatic interactions were detected between substrates (linoleic acid and arachidonic acid) and mutated PLAT/LH2 domain (lacking lysine) of Lox-2 as compared to strong interactions of the substrates with domain of the wild type. This is ascribed to the formation of two hydrogen bonds between positively charged lysine (K119) and negatively charged lipid substrates in the latter and the absence of hydrogen bonds at the corresponding site in the mutant. The strong interaction is essential for bringing the isozyme active site into close association with its substrate and this failure in the mutant led to reduction of isozyme activity in T2 seeds. The present study is distinct from Wang et al. (2020) for structural modelling analysis. Li et al. (2013) suggested that the charge carried by lysine enables it in establishing strong electrostatics interaction.

Lipoxygenases are non-heme iron enzymes, and targeting of histidine (H532), an iron-binding ligand in the lipoxygenase domain displayed complete dysfunctional Lox-2 protein (Wang et al. 1994; Reinprecht et al. 2011). In present study, the conversion of lysine (K119) in PLAT/LH2 domain resulted in reduction of lipoxygenase isozyme activity, and lysine is also an iron-binding ligand essential for the enzymatic activity (Clydesdale 1982). The elimination of seed lipoxygenases through dysfunctional Lox-2 protein could produce better quality soy-products (Reinprecht et al. 2011). However, the enzymes of Lox pathway are associated with diverse plant growth and developmental processes, such as lipid metabolism, nitrogen storage, seed germination, plant–microbe interactions, resistance against various abiotic and biotic stresses etc. (Porta and Rocha-Sosa 2002). As the complete dysfunction of Lox proteins could disrupt plant physiological processes, therefore in the present study, reduction in Lox-2 isozyme activity was intended, rather than complete dysfunction through mutation in PLAT/LH2 domain of a commercial soybean cultivar SL1074.

The mutation in Lox-2 was stably transmitted to succeeding generations. The mutants with reduced lipoxygenase isozyme activity will be characterized for agronomic performance, resistance against various abiotic, biotic stresses and undesirable flavour in seeds. The mutant soybean with reduced isozyme activity will be an important source for introgressing the trait in plant breeding programs.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (PDF 2915 KB) (2.8MB, pdf)

Acknowledgements

The authors are grateful to School of Agricultural Biotechnology for providing financial and infrastructural support to carry out this research.

Author contributions

EP, PD, SH executed the experiments and collected the data. JSS, EP, AK, GC, SS, BSG analyzed and interpreted the data. JSS conceptualized, designed the research work and prepared the manuscript. All authors reviewed the manuscript.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Conflict of interest

The authors declare no competing interests.

Consent for publication

All authors consent to publication of this article.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors. The study was reviewed and approved the Punjab Agricultural University Institutional Biosafety Committee.

Footnotes

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Supplementary Materials

Supplementary file1 (PDF 2915 KB) (2.8MB, pdf)

Data Availability Statement

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