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. 2020 Apr 13;18(11):2241–2250. doi: 10.1111/pbi.13380

Gene editing of three BnITPK genes in tetraploid oilseed rape leads to significant reduction of phytic acid in seeds

Niharika Sashidhar 1, Hans J Harloff 1, Lizel Potgieter 2,3, Christian Jung 1,✉
PMCID: PMC7589381  PMID: 32191373

Summary

Commercialization of Brassica napus. L (oilseed rape) meal as protein diet is gaining more attention due to its well‐balanced amino acid and protein contents. Phytic acid (PA) is a major source of phosphorus in plants but is considered as anti‐nutritive for monogastric animals including humans due to its adverse effects on essential mineral absorption. The undigested PA causes eutrophication, which potentially threatens aquatic life. PA accounts to 2‐5% in mature seeds of oilseed rape and is synthesized by complex pathways involving multiple enzymes. Breeding polyploids for recessive traits is challenging as gene functions are encoded by several paralogs. Gene redundancy often requires to knock out several gene copies to study their underlying effects. Therefore, we adopted CRISPR‐Cas9 mutagenesis to knock out three functional paralogs of BnITPK. We obtained low PA mutants with an increase of free phosphorus in the canola grade spring cultivar Haydn. These mutants could mark an important milestone in rapeseed breeding with an increase in protein value and no adverse effects on oil contents.

Keywords: Brassica napus, BnITPK, CRISPR‐Cas, inositol (1, 3, 4) P3 5/6 Kinase, inorganic phosphorus, lpa mutants, phytic acid, polyploidy, rapeseed meal

Introduction

Seeds are major reserves for nutritional elements. Inositol hexakisphosphate, also known as phytic acid (PA), contributes to 65‐90% of total phosphorus in seeds across a wide range of plant species from cereals to oilseeds (Raboy, 2000). PA is a negatively charged molecule and chelates essential minerals, thereby leading to the so‐called hidden hunger (Gibson et al., 2018; Perera et al., 2018). Furthermore, due to lack of phytases, monogastric animals including humans cannot digest PA and the undigested PA is causing eutrophication (Raboy et al., 2000; Shi et al., 2007). In plants, PA is synthesized in two different ways, via a lipid‐dependent and a lipid‐independent pathway (Raboy, 2003). The PA pathway is initiated by oxidative cyclization of glucose 6‐phosphate to myo‐inositol monophosphate via myo‐inositol phosphate synthase. Subsequently, myo‐inositol monophosphate is converted to free myo‐inositol via de‐phosphorylation by myo‐inositol monophosphatase. Concurrently, a reverse reaction occurs by myo‐inositol kinase, where the free myo‐inositol is phosphorylated again to myo‐inositol monophosphate. These reactions are considered to be the housekeeping pathway for various myo‐inositol‐dependent pathways and commonly known as Loewus pathway (Donahue et al., 2010; Loewus and Murthy, 2000). Furthermore, interaction of myo‐inositol with phosphatidyl lipids leads to the lipid‐dependent, whereas subsequent phosphorylation by respective kinases leads to the lipid‐independent pathway (Figure 1) (Raboy, 2009). Although both pathways contribute to PA biosynthesis, the lipid‐independent pathway is considered as the predominant pathway for PA accumulation in seeds (Raboy, 2000). Finally, the synthesized PA is transported by a multidrug‐resistant protein into vacuoles where it is stored as calcium, magnesium and potassium salts referred to as phytins (Figure 1) (Otegui et al., 2002). Upon seed germination, endogenous phytases are activated and PA is hydrolysed to free inorganic phosphate (Pi) and myo‐inositol (Yao et al., 2012). The released Pi and partially phosphorylated inositols are utilized in various physiological processes during plant development including DNA repair, mRNA export, cellular signalling, and biotic and abiotic stress tolerance (Dieck et al., 2012; Sparvoli and Cominelli, 2015). In oilseed rape, PA contents vary from 2 to 5%. This includes quality type (‘canola’) varieties with reduced erucic acid and glucosinolate contents (Tan et al., 2011; Thompson, 1990; Uppström and Svensson 1980; Zhao et al., 2008). So far, approaches to lower the PA content in extracted rapeseed meal focused either on adding phytases to the meal or to remove PA by technical processes (Adem et al., 2014; El‐Batal and Abdel Karem 2001). In spite of these strategies to reduce PA, there are still several obstacles for an efficient purification of the meal (Dersjant‐Li et al., 2015). In this study, we aimed to knock out the key enzyme ITPK (inositol tetrakisphosphate kinase), which is catalysing the penultimate step for the synthesis of PA in plants (Raboy, 2009). ITPK is a highly conserved enzyme across various species from plants to humans and has been shown to have diverse functions (Jiang et al., 2019). It belongs to the super family of ATP‐grasp fold proteins and is involved in photomorphogenesis by interacting via the COP9 signalosome, in stress responses, in signal transduction and in seed coat development (Du et al., 2011; Qin et al., 2005; Sweetman et al., 2007; Tang et al., 2013). In our previous study, we have identified EMS (ethyl methanesulphonate)‐induced loss‐of‐function mutations in six genes of the PA biosynthesis pathway (Sashidhar et al., 2019). However, due to gene redundancy in polyploid rapeseed noticeable phenotypic effects could only be observed in plants carrying multiple mutations which required time‐consuming crossing experiments. Thereby, functional analysis of traits that are encoded by several paralogs needs a fast forward approach for simultaneous knockouts. In this study, we aimed to knock out multiple copies of the key enzyme ITPK (inositol tetrakisphosphate kinase), which is catalysing the synthesis of inositol pentakisphosphate by implementing CRISPR‐Cas9‐mediated mutagenesis (Figure 1) (Zhang et al., 2012), to obtain lpa mutants with no pleiotropic effects. Therefore, our study offers new perspectives for breeding low PA rapeseed.

Figure 1.

Figure 1

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Proposed phytic acid pathway in plants (modified after Raboy et al (2009)). The grey colour box indicates the Loewus pathway, which is a housekeeping pathway for various myo‐inositol‐dependent pathways. Blue and red arrows indicate the lipid‐independent pathway and lipid‐dependent pathway, respectively. The targeted gene (ITPK) for CRISPR‐Cas9 mutagenesis is highlighted in green. MIPS: myo‐inositol phosphate synthase, MIK: myo‐inositol kinase, IMP: myo‐inositol monophosphatase, 2‐PGK2: 2‐phosphoglyceric acid kinase, ITPK: inositol tetrakisphosphate kinase, IPK1: inositol pentakisphosphate 2‐kinase, IPK2: inositol multiphosphate kinase, MRP5: multidrug resistance protein, DAG: diacyl glycerol, PLC: phospholipase C, PtdIS: phosphatidyl inositol synthase.

Results

Identification of ITPK genes and selection of paralogs for mutagenesis

So far, no oilseed rape orthologs of Arabidopsis thaliana ITPK genes have been identified. We have used four known AtITPK sequences for a BLAST query in the B. napus database (http://www.genoscope.cns.fr/brassicanapus/) which resulted in 15 paralogs (Table 1). The gene families in B. napus were referred to as BnITPK1 to BnITPK4, and the homology between members of the same gene family ranged between 89 and 99%. BnITPK1 shared approx. 80% similarity with BnITPK2 and BnITPK3, but less than 20% similarity with BnITPK4 (Table 1). In order to find suitable target genes for a CRISPR‐Cas9 knockout experiments, a phylogenetic tree was constructed between BnITPKs and ITPK orthologs from other plants. The analysis resulted in three distinct clustered α, β and γ groups (Figure 2). Typically, ITPK orthologs which had been successfully targeted for mutations in previous studies belonged to the α and γ groups (Sparvoli and Cominelli, 2015). We expected that knocking out the orthologs in rapeseed would give comparable results and therefore focused on the α and γ group genes BnITPK1 and BnITPK4 (Figure 2). The BnITPK1 and BnITPK4 genes could be amplified by PCR in the cv. Haydn, with the exception of Bn.ITPK4.C04a (Table S1). However, several primer combinations failed to amplify the putative Bn.ITPK4.C04a ortholog from five different rapeseed genotypes (cv. Haydn, cv. Express 617, cv. Mozart, cv. Tapidor and RS 306) which was therefore excluded from further studies. For CRISPR‐Cas9 knockout studies, we choose to knock out paralogs of BnITPK1 and BnITPK4 gene families in a single plant.

Table 1.

Features of BnITPK genes used in this study

Arabidopsis gene B. napus sequence annotation B. napus paralogs Genomic sequence length (bp) Exon/intron structure Coding region (bp) Protein size (aa) Genome sequence identity with AtITPK (%) Amino acid sequence identity with AtITPK (%)
Exons Introns
AtITPK1 BnaA10g17710D Bn.ITPK1.A10 960 1 0 960 320 86.7 89.3
BnaC09g41080D Bn.ITPK1.C09 960 1 0 960 320 86.1 89.3
BnaC03g07940D Bn.ITPK1.C03 975 1 0 975 325 84.8 88.4
BnaA03g06170D Bn.ITPK1.A03 981 1 0 981 327 84.9 88.4
AtITPK2 BnaAnng34680D Bn.ITPK2.Ann 2454 9 8 1164 388 89.2 84.5
BnaC01g04480D Bn.ITPK2.C01 2456 9 8 1155 385 89.3 85.4
BnaA03g50630D Bn.ITPK2.A03 2316 9 8 1158 386 88.5 84.9
BnaC07g44490D Bn.ITPK2.C07 2391 9 8 1185 395 88.5 82.1
BnaC03g66400D Bn.ITPK2.C03 2539 10 9 1041 347 89.7 82.3
BnaA01g03220D Bn.ITPK2.A01 969 5 4 654 218 88.8 83.1
AtITPK3 BnaCnng47190D Bn.ITPK3.Cnn 1964 9 8 1077 359 88.8 80.3
BnaA03g38640D Bn.ITPK3.A03 1451 8 7 930 310 82.6 80.5
AtITPK4 BnaA05g03660D Bn.ITPK4.A05 2634 12 11 1467 489 88 86.9
BnaC04g03240D Bn.ITPK4.C04a 2455 12 11 1374 458 88.7 86.7
BnaC04g03250D Bn.ITPK4.C04b 2751 12 11 1452 484 89 86.7
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Figure 2.

Figure 2

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Phylogenetic tree of ITPK genes in different plant families. The tree was constructed using the maximum likelihood method with nucleotide substitution model as general time reversible. Bootstrapping using 500 replicates. At – A. thaliana, Bn – B. napus, Os – Oryza sativa, Gm – Glycine max, Zm – Zea mays, Hs – Homo sapiens, Eh – Entamoeba histolytica. Accession numbers of rice ITP5/6Ks are as follows: OsITPK5/6‐1 (AK106544), OsITPK5/6‐2 (AK100971), OsITPK5/6‐3 (AK067068), OsITPK5/6‐4 (AK071209), OsITPK5/6‐5 (AK059148) and OsITPK5/6‐6 (AK102571) (Kim and Tai, 2014).Arabidopsis and B. napus genes are mentioned in the Table 1. Eh: AF118848, Hs: NP055031, Gm: type 1 – EU033958, type 2 – EU033959, type 3 – EU03396h, type 4 – EU033961, Zm: AY172635 (Stiles et al., 2008). The groups of ITPKs are shown as α, β and γ.

CRISPR‐Cas9 mutagenesis of BnITPK genes

Due to different gene structures between BnITPK1 and BnITPK4, two different target sites, referred to as target sites 1 and 2, respectively, were chosen (Figure 3). Within each family, the target sequences differed by one nucleotide between the respective members of the family (position 10 upstream of the PAM sequence) (Figure 3). Since the BLAST search against the rapeseed reference genome did not show any off‐targets, neither in BnITPK2 and BnITPK3 genes nor in any other coding sequence of the rapeseed genome, these target sites were used for subsequent gene editing. Moreover, the genes containing the SNPs were lowly expressed in developing seeds of Express 617 and leaves of a Chinese semi‐winter line compared to the other members of the respective family (Shah et al. 2018) (Figure S1), indicating that they might play a minor role in PA biosynthesis in oilseed rape.

Figure 3.

Figure 3

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Gene structure and CRISPR‐Cas9 target sites in six BnITPK genes. Transcribed sequences of BnITPK1 and BnITPK4 gene families. Grey and green boxes indicate the 5’ UTR and 3’ UTR, respectively. Open boxes indicate exons, and the black lines indicate the introns. Blue boxes indicate target sites. The Bn.ITPK1.A10 sequence represents target site 1, whereas the Bn.ITPK4.C04b sequence represents target site 2 (Express 617). SNPs are indicated by blue letters. The Streptococcus pyogenes PAM sequence is underlined.

Hypocotyl transformation was performed to introduce the CRISPR‐Cas9 constructs into the spring cv. Haydn. A total of 321 hypocotyls were co‐cultivated with Agrobacterium tumefaciens containing the Cas9‐sgRNA cassette with both target sites. From the treated hypocotyls, 23 shoots from 10 independent transgenic events (termed T1 generation) were regenerated to whole plants (itpk_C1 to itpk_C10) on BASTA selection media (Table S2). All T1 plants were tested positive for the presence of the transgene using primers flanking the Cas9 gene (Table S1), which is corresponding to a transformation efficiency of 3.1%. PCR amplicons from leaf DNA of each transformant were sequenced to search for mutations within the targeted regions. All T1 plants showed gene editing in up to four genes. Three mutants were selected for further studies (itpk_C2.1, itpk_C3.5 and itpk_C6.1). Sanger sequencing revealed an overlay of more than two different sequences, suggesting that these were chimeric plants and that some mutations might have occurred somatically. Therefore, amplicons containing the target regions were cloned into plasmids and were subsequently sequenced. We observed the presence of three to eight different mutant alleles (single letter code A, B, E, G) for each gene along with non‐edited alleles for each of the genes targeted thus confirming the chimeric nature of the T1 plants (Figure 4).

Figure 4.

Figure 4

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Pedigree of three BnITPK mutant generations derived from three different T1 plants. Target sites and T‐DNA insertion sites were genotyped with primer combinations given in Table S. Each box represents the mutated and non‐mutated alleles in a single plant. Red boxes indicate transgenic, and black boxes indicate non‐transgenic plants, respectively. A non‐edited Haydn allele is indicated by a suffix ‘h’, whereas edited alleles are numbered according to their mutation type (see Table S3). Letter codes A, B, E and G indicate alleles of Bn.ITPK1.A10, Bn.ITPK1.C09, Bn.ITPK4.A05 and Bn.ITPK4.C04b, respectively. Homozygous plants used for phenotyping were obtained in the T4 generation. Seed codes are written on top of each genotype. Dotted arrows indicate progenies that were not further analysed in this study. The number of plants used for genotyping is given in blue colour.

Generating homozygous double and triple mutants

We expected that chimeric T1 plants give rise to complex segregation patterns in the T2 generation and that homozygous mutants can only be selected from later generations. Of each T2 population, 10 (itpk_C2.1 and itpk_C6.1) and 55 plants (itpk_C3.5), respectively, were genotyped with locus‐specific primers (Figure 4). All T1 mutant alleles were found in the T2 generations except in the itpk_C3.5 offspring where not any of the Bn.ITPK4.A05 mutations (E1 , E2 , E3 and E4 alleles) was inherited to the next generation. We reason that these mutations were absent from the germ‐line; thus, they were not transmitted to the next generation. This was also consistent with the T1 plasmid sequencing results where the ratio of edited to non‐edited alleles was considerably low. Interestingly, new mutant alleles appeared in two T2 populations (offspring of itpk_C2.1 and itpk_C6.1) which had not been detected in their T1 parents (Figure 4). Furthermore, complex editing patterns were found even in T2 plants illustrating that Cas9 activity giving rise to de novo mutations in this generation. However, no editing was observed in Bn.ITPK1.A03 and Bn.ITPK1.C03 (Ch and Dh ). The presence of a transgene in all analysed T2 plants suggested that more than one copy of the transgene was inserted which explains that Cas9 was still active in all T2 plants analysed. Since the mutant alleles were not fixed in T2 plants, three genotypes from each family (seed codes: 180716,180663 and 180705) were randomly chosen and selfed to get homozygous non‐transgenic mutants in the T3 generation. Firstly, the segregation ratios for the transgene loci were determined. T3 offspring of itpk_C2.1, itpk_C3.5 and itpk_C6.1 showed a 3:1 segregation (transgenic: non‐transgenic) indicating a single insertion event (Table S4). Secondly, homozygous double mutants (BnITPK1) and homozygous triple mutants (BnITPK1 and BnITPK4) were identified in the T3 generation. Heterozygous triple mutations for BnITPK1 and BnITPK4 genes were found in the itpk_C3.5 progenies (Figure 4, Table S3). Finally, T4 populations were produced where non‐transgenic homozygous plants for the ITPK mutations were selected for phenotypic analysis.

Triple mutants showed reduced PA contents and increased Pi levels

Four T4 mutant lines homozygous for the mutations were chosen to examine the effect of the edited alleles. Two triple mutants (Nos. 190665 and 190666) with different Bn.ITPK1.C09 mutant alleles and two double mutants (Nos. 190667 and 190669) with the same mutated BnITPK1 alleles were analysed. A T4 line homozygous for the Haydn alleles (190668) and the donor line Haydn were used as controls (Figure 5a). Only the two triple mutants displayed significantly reduced (27.2% ‐ 35.3%) seed PA contents, while the reduction in double mutants was not significant as compared to the control plants (Figure 5b). Furthermore, no partially phosphorylated inositol phosphate intermediates could be detected. To find out whether a decreased amount of PA led to an increase of Pi contents in the seeds, Pi contents were measured in all the genotypes with the colorimetric ammonium molybdate test. As expected, the two triple mutants showed higher Pi contents as compared to the control. These results demonstrate that in oilseed rape, a decrease of PA is highly correlated with an increase of free phosphorus (Figure 5b). However, this increase is not molar equivalent to the reduced PA contents, which might indicate that less phosphorus is taken up by the plants.

Figure 5.

Figure 5

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Genotypes and phenotypes of BnITPK mutants. (a): A non‐edited allele is indicated by a suffix ‘h’, while edited alleles carry numbers (see Table S3). WT stands for the Haydn allele (wild type). CRISPR‐Cas9 mutations are indicated by red letters (insertions) and by ‘‐’ (deletions). (b): Phytic acid and free inorganic P were measured in homozygous T5 seeds. ANOVA and post hoc test using Tukey’s multiple comparison test; p = 0.05 were performed for statistical significance. Similar letters indicate no significant differences.

We questioned whether the CRISPR‐Cas9 mutations could have pleiotropic effects on yield‐related traits and seed vigour. We observed that mutant plants grew normally and did not show any obvious phenotype. Only minor differences were observed between mutant and control lines. Plant height and thousand kernel weight varied slightly between mutants. The vigour of T5 seedlings was evaluated five days after germination. Root length and hypocotyl length did not vary significantly as compared to the controls. The seed oil content, a major yield component of B. napus, was between 40 and 50%, which is within the range of modern rapeseed cultivars (Figure 6). Although the experiments were conducted under controlled conditions, the data suggest that altered PA and Pi contents might not have a negative impact on important agronomic traits in rapeseed.

Figure 6.

Figure 6

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Yield‐related traits of BnITPK double and triple mutants. Plants were grown in the greenhouse under long day conditions (16h light/8h dark) at 22°C, and T5 seeds were analysed. Line 180800 is a mutant offspring carrying the Haydn alleles. Root and hypocotyl length were measured five days after germination. The oil content was measured using the n‐hexane method. Statistical significance was calculated using ANOVA followed by a post hoc test (Tukey) in R. 3.6.1. Same letters indicate no significant effect. + indicates the mean of the samples.

Discussion

This study describes low phytic acid mutants in oilseed rape by targeting genes from the PA biosynthesis pathway. We selected the BnITPK gene family as a suitable target for knockout analysis by CRISPR‐Cas9 in the canola grade cv. Haydn. We found a reduction of about 35% of PA with a simultaneous increase of Pi by knocking out three essential BnITPK genes. Interestingly, a significant reduction in PA content was only observed in triple mutants. This clearly demonstrates that knockout of multiple paralogs is necessary to observe a noticeable phenotype which can only be achieved by CRISPR‐Cas mutagenesis. Our data are in line with previous studies in wheat and potato where only multiple knockouts of different paralogs resulted in a desired phenotype (Andersson et al., 2017; Wang et al., 2014; Zhang et al., 2019). In rapeseed, the situation is even more complicated because during evolution, the genome underwent several whole‐genome duplication and triplication events resulting in an average copy number of two to eight for each Arabidopsis ortholog (Chalhoub et al. 2014). Another important aspect of our study adds to our understanding of CRISPR‐Cas mutations during plant development and between generations. We observed mutations in T2 generation that had not been found in their T1 parents. One reason could be that only a small sector of the T1 plant carried the mutation, which was not detected by sequencing. However, we favour another explanation that de novo mutations appeared in the T2 generation. This could be due to the low activity of the Cas9 gene which is influenced by the position of the T‐DNA in the genome or by multiple T‐DNA insertions which can result in low transcriptional activities or even gene silencing (De Buck et al., 2009; Tang et al., 2006). It is also worth mentioning that in this study, Cas9 acted specifically and did not tolerate SNPs in the target site for inducing mutations, which is in contrast to our experiments performed on BnALC genes in B. napus (Braatz et al., 2017). In conclusion, future studies should carefully examine also later generations even if T1 plants were lacking mutations.

Our study adds to our understanding of PA biosynthesis and storage in rapeseed. We demonstrate that ITPKs play an important role in PA metabolism and thus are a suitable target for reducing PA amounts. The ITPK gene was first identified in maize lpa2 mutants (zmitpk) which showed a 30% reduction in PA without any effect on plant performance. Subsequently, knockout mutants with reduced PA contents in seeds have also been described in rice, Arabidopsis and maize (Raboy, 2009; Shi et al., 2003). A study in rice identified two knockout mutants of OsITPK6 showing 46% reduction of PA seed content caused by a mis‐sense mutation and 68% caused by a splice site mutation without any adverse effects on seedling performance (Kim and Tai, 2011). Another study in rice showed contradictory result. While the PA content was reduced by 32% due to the loss of two amino acids in the OsITPK6 protein, negative effects on seed set, seed weight and germination were reported (Jiang et al., 2019). Mutants in Arabidopsis atitpk1 and atipk4 showed a 46% and 51% reduction, respectively, with no pleiotropic effects on seed germination and seedling performances (Kim and Tai, 2011). We also did not observe any negative effects on germination rate and seedling vigour which suggests that PA reduction in our mutants is below a critical threshold. We observed that the triple mutant with a 6‐bp deletion (line No. 190666) had the lowest PA content. According to protein modelling, this mutation results in the loss of two amino acids within an ATP‐binding site (see Figure S2, Figure S3) which has also been proposed for a StITPK1 ortholog in potato (Caddick et al., 2008). Although the contribution of each gene to the synthesis of PA in B. napus is unknown, we reason that BnITPK genes act in an additive manner and that an even higher reduction could be achieved by a complete knockout of all BnITPK1 and BnITPK4 genes. It is important to know what happens to inorganic seed Pi if it is not bound in PA. Due to the loss of PA, the triple mutants showed a threefold increase of Pi. Similar results were obtained with a maize lpa2 mutant where the Pi content was increased by threefold (Shi et al., 2003). The lower increase of Pi was also shown previously as a general pattern of itpk mutants in rice and Arabidopsis with decreased contents of PA accompanied by a minor increase of Pi and lower inositol phosphates (Sparvoli and Cominelli, 2015). It is noteworthy that in our study, the increase in seed Pi was also not a molar equivalent to the reduced PA contents, which might indicate that less phosphorus is needed by the mutants.

Lowering the PA amounts in seeds is an important milestone for enhancing rapeseed meal quality for human and animal diets. Moreover, the mutants offer new perspectives for oilseed rape breeding. 1) Reduced PA content will be beneficial to combat the mineral deficiencies of the meal. 2) Recovery of rapeseed protein will be more efficient due to lower the amount of tightly bound PA. 3) Protein contents will be improved with no effect on oil contents and thus overruling their negative correlation (Jasinski et al., 2018). 4) Increase of Pi contents might pave ways to reduced dependency on phosphate fertilizers, which is a non‐renewable resource (Cordell et al., 2009). 5) Increased bioavailability of Pi attenuates eutrophication of waters (Conley et al., 2009). B. napus is primarily an oil crop but can also be used as a nutritional source due to its rich protein and well‐balanced amino acid contents for human and animal diets (Campbell et al. 2016; Wanasundara et al., 2016). There is increasing effort to improve the nutritional value of B. napus meal in order to overcome the dependence on the expensive soybean imports in regions where soybean cannot be grown (Jasinski et al., 2018). So far, rapeseed meal is used for aquatic and poultry industries as a feed, but it can also be used as a valuable protein source for humans and non‐ruminants (Gacek et al., 2018). However, the presence of various anti‐nutritive compounds like phytic acid, sinapine, fibre and glucosinolates impedes its commercialization as a major protein source. While rapeseed cultivars and lines exist which are low in glucosinolates, sinapic acid and erucic acid, no reduced PA lines are available. Furthermore, the protein extraction from seeds is impaired by the presence of tightly bound phytins and tannins (Wanasundara et al., 2016), which reduces the digestion by enzymes like trypsin and pepsin but also inhibits α‐amylases at physiological pH in monogastric animals (Reddy et al., 1996). Therefore, the established mutants pave the way to breed rapeseed with improved meal quality and reduced phosphorous demand.

Experimental procedures

Plant material and growth conditions

The German spring cultivar Haydn (double low ‘00’ or ‘canola’ quality) was used for transformation experiments. Greenhouse experiments were performed under long day conditions (16h light and 8h dark) at 22°C. Plants were grown in 11x 11cm pots, and they were not fertilized to avoid phosphorous contamination. Upon flowering, the inflorescences were bagged, and dry mature seeds were harvested. For phenotyping experiments, five biological replicates for each genotype were grown. Plants were randomized every week, and plant height (length between base of the stem to the tip of the inflorescence) and thousand kernel weight (TKW) were measured at the time of harvesting. Seed oil content was measured using the n‐hexane method. To measure the seedling’s vigour, 20 seeds of each genotype were sown on ½ Murashige–Skoog (MS) medium and germinated in the dark. Five days after germination, the root and hypocotyl lengths were measured.

Phylogenetic analysis

Published ITPK sequences were retrieved from respective crop’s databases. Sequences from rice were obtained from http://rice.plantbiology.msu.edu/cgi‐bin/gbrowse/rice/#search , Arabidopsis from TAIR (https://www.arabidopsis.org/servlets/Search?action=new_search&type=gene), and soybean, maize and human sequences were retrieved from NCBI (https://www.ncbi.nlm.nih.gov/). The amino acid sequences of the ITPK proteins were aligned using MAFFT v7.313 with the G‐INS‐i strategy (Katoh and Standley, 2013). The alignment was manually curated. The substitution model for the maximum likelihood analysis of the curated alignment was determined by ProtTest v3.4.2 (Abascal et al., 2005). The maximum likelihood phylogenetic analysis was conducted with MEGA X (Kumar et al., 2018). A JTT model was applied, and 500 bootstrap replicates were performed. The resulting phylogenetic tree was visualized with iTOL (Letunic and Bork, 2007; Letunic and Bork, 2019).

Target site design and plasmid vector construction

The vectors pChimera and pCas9_TPC were obtained from Prof. Holger Puchta (Karlsruhe institute of technology, Germany). pChimera contains the sgRNA sequence driven by an Arabidopsis thaliana U6‐26 promoter and pCas9_TPC vector has the Cas9 gene from Streptococcus pyogenes driven by an Ubi4‐2 promoter from parsley. These vectors have ampicillin and spectinomycin resistance genes, respectively, as selective markers for bacteria and BASTA resistance to select transgenic shoots (Fauser et al., 2014).

The BnITPK CRISPR‐Cas9 cassette was constructed according to published protocols (Fauser et al., 2014) with minor modifications. sgRNAs targets were cloned into the pCas9‐TPC vector by using AvrII and BcuI restriction enzymes, as these are isocaudomeres. The final sgRNA‐target vector was transformed into the Agrobacterium tumefaciens strain GV3101 pMP90RK, which was then used for rapeseed hypocotyl transformation.

We used four Arabidopsis sequences encoding ITPK genes to BLAST against the rapeseed reference genome (http://www.genoscope.cns.fr/brassicanapus/) (Kim and Tai, 2011). Retrieved sequences were annotated according to the Genoscope browser using the CLC Main Work Bench 7.9.1 (CLC, Aarhus, Denmark) software. For the target design, all paralogs of each ITPK gene family were aligned and regions of 20bp length and a GC content> 40% were chosen which were next to a PAM sequence (NGG). Conserved regions with a maximum of one SNP upstream of 10th position from the PAM site were considered as suitable targets because they can be tolerated by Cas9 (Braatz et al., 2017). All target sites chosen were blasted against the B. napus genome database using CLC Main Work Bench 7.0.3 for verification of any off‐targets. Sequences lacking BLAST hits were selected for further analysis. Oligonucleotides were ordered from Eurofins Genomics Company (Ebersberg, Germany) and purified by a high purity salt free (HPSF) method with a purity of> 70 %. (http://www.eurofins.de/de‐de.aspx).

Rapeseed hypocotyl transformation

We used a protocol kindly provided by Dr. José Orsini (Saaten‐Union Biotec GmbH, Germany) with minor adaptations from a published protocol (Zarhloul et al. 1999). Seeds from cultivar Haydn were surface sterilized with 70% EtOH for two minutes followed by 3% sodium hypochlorite and 0.01% Tween 20 for 10 min. Seeds were washed thoroughly with autoclaved water and sown on MS medium. Five‐day‐old hypocotyls were used for transformation. The hypocotyls were cut into 1cm explants and incubated for 45 to 60 min with an A. tumefaciens overnight culture adjusted to OD600 1.2. The hypocotyls were co‐cultivated for two days and transferred to shoot‐inducing medium containing 500 mg L‐1 ticarcillin disodium/clavulanate potassium (Duchefa‐T0190.0002). After 4 weeks on shoot‐inducing medium, the regenerated shoots were transferred to shoot‐inducing medium containing 5 mg L‐1 BASTA (phosphinothricin) (Duchefa: P0159.0250). Surviving shoots were transferred to root‐inducing medium for regeneration of complete plants.

The transformation efficiency was calculated as (number of independent transgenic plants/ total number of explants used) * 100.

Verification of the mutations by molecular marker analysis

Paralog‐specific primers were designed using the Darmor‐bzh reference sequence to amplify the individual copies according to the protocol (Table S) (Liu et al., 2012). DNA was isolated from the leaf samples using the CTAB method. Sanger sequencing was performed from the paralog‐specific amplicons to verify the putative editing events. Furthermore, PCR amplicons from chimeric T1 plants were cloned into the pGEM‐T vector (Promega, Germany) and plasmids were Sanger‐sequenced. For verification of the underlying alleles in the chimeric plants in the T2 generation, we used an online software tool called Tracking of InDels by Decomposition (TIDE) (https://tide.deskgen.com/) (Brinkman et al., 2014). Raw Sanger sequencing reads of individual paralogs (.abi files) were used as query, which were then compared to the provided reference sequences (non‐edited sequences). The analysis resulted in plots for each sequence of a gene showing the probable nucleotide changes caused by the NHEJ repair mechanism.

Measuring seed phytic acid and inorganic phosphorous contents

Phytic acid and inorganic phosphorus (Pi) were extracted following the procedure of (Matthäus et al., 1995) with slight modifications. Dry matured seeds of 200 mg were ground into fine powder from five biological replicates and from each sample, three technical repeats of ~ 50 mg were analysed. Defatting was done by extraction with 1 ml n‐hexane overnight at room temperature on an overhead shaker. After centrifugation (10 min at 20,800 g), the defatted seed cake was extracted with 1 ml of 0.5 M HCl by vigorous shaking on a Bioer mixing block (MB‐102) at 30°C for 4h. After centrifugation at 20,800 g, the supernatant was freeze‐dried and re‐dissolved in 1 ml double distilled water. A 0.5‐ml aliquot was applied on a 2 ml column of Dowex 1x2 (Acros Organics, A0346424), washed with 2x10 ml water and eluted with 25 ml 2 M HCl. The eluates were freeze‐dried and re‐dissolved in 1 ml double distilled water. Phosphoinositols (IP1 to IP6) were analysed by HPLC following the procedure published by (Rounds and Nielsen, 1993) with minor modifications. Fifty‐microlitre samples were injected onto a PL‐SAX 1000 HPLC column (50 x 4.6 mm, 5µ, Agilent), and IP1 to IP6 were eluted at 30°C with a flow rate of 1 ml min‐1. A 25‐min linear gradient from 0.01 M 1‐methylpiperazine (pH 4.0) to 0.5 M NaNO3 dissolved in 0.01 M 1‐methylpiperazine (pH 4.0) with a further elution for 2 min, 1 min returning to starting conditions and 10 min equilibration was used. Analytes were detected at 500 nm using a Pinnacle PCX device (LCTech, Germany) with a post‐column reagent consisting of 0.015% (w/v) FeCl3 x 6H2O and 0.15% (w/v) sulphosalicylic acid. A calibration curve was plotted using 10‐50 µg of PA sodium salt hydrate (Sigma: P8810). For determining Pi contents, 50µl of the purified column extracts were mixed with 500 µl of colouring reagent (10% w/v of ascorbic acid (Roth‐ Art.Nr.3666.1) and 5% w/v of ammonium molybdate (Roth, Art. No.3525.2), adjusted to 1.5 ml with double distilled water and incubated at 40°C for one hour. The samples were measured against a reagent blank in a spectrophotometer at 655 nm. A calibration curve 0 to 100 nmol Pi was used.

Statistical analysis

For greenhouse experiment, five plants of each line were grown (biological replicates). Of each plant, three technical replicates were taken for PA, inorganic P and oil measurements. An ANOVA was performed using R 3.6.0 and the MulticomView package, and post hoc test was done by using Tukey’s multiple comparison test; p = 0.05.

Conflict of interest

The authors have no conflict of interest in relation to this work.

Author contributions

N.S planned, performed and analysed the experiments and wrote the article; L.P. performed the phylogenetic analysis; H.‐J.H. and C.J. designed the study and supervised the experiments; and H.‐J.H and C.J. revised the manuscript. All authors read and approved the final manuscript.

Supporting information

Figure S1 Expression analysis of BnITPK paralogs in seeds and leaves.

Figure S2 Protein alignment of ITPK showing the inositol, ATP and Mg2+ binding sites.

Figure S3 Predicted protein structure of Bn.ITPK1.C09 in Haydn and in the mutant line 190666.

Table S1 Primer sequences used in this study.

Table S2 Editing status in regenerated T1 plants.

Table S3 Gene acronyms for four BnITPK genes used in this study.

Table S4 Segregation analysis in T3 generations.

PBI-18-2241-s001.pdf (1.3MB, pdf)

Acknowledgements

We thank Dr. Siegbert Melzer for his valuable discussions and Birgit Defant and Ines Schütt for helping with the tissue culture and greenhouse experiments. We also thank the Institute of Clinical Molecular Biology in Kiel for providing Sanger sequencing as supported in part by the DFG Clusters of Excellence ‘Precision Medicine in Chronic Inflammation’ and ‘ROOTS’ and thank T. Naujoks, Dr. D. Langfeldt and Dr. B. Löscher for technical support. We thank Dipl. Biol. Jens Hermann and Prof. Dr. Wolfgang Bilger from the Department of Ecophysiology of Plants at the CAU Kiel for HPLC measurements and Prof. Holger Puchta from KIT, Karlsruhe for supplying the CRISPR vectors. This study was supported by a grant from the German Research Foundation, DFG (Grant No. JU 205/26‐1).

Sashidhar, N. , Harloff, H. J. , Potgieter, L. and Jung, C. (2020) Gene editing of three BnITPK genes in tetraploid oilseed rape leads to significant reduction of phytic acid in seeds. Plant Biotechnol. J., 10.1111/pbi.13380

References

  1. Abascal, F. , Zardoya, R. and Posada, D. (2005) ProtTest: selection of best‐fit models of protein evolution. Bioinformatics (Oxford, England) 21, 2104–2105. [DOI] [PubMed] [Google Scholar]
  2. Adem, H.N. , Tressel, R.‐P. , Pudel, F. , Slawski, H. and Schulz, C. (2014) Rapeseed use in aquaculture. OCL 21, D105. [Google Scholar]
  3. Andersson, M. , Turesson, H. , Nicolia, A. , Fält, A.‐S. , Samuelsson, M. and Hofvander, P. (2017) Efficient targeted multiallelic mutagenesis in tetraploid potato (Solanum tuberosum) by transient CRISPR‐Cas9 expression in protoplasts. Plant Cell Rep. 36, 117–128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Braatz, J. , Harloff, H.J. , Mascher, M. and Stein, N. (2017) CRISPR‐Cas9 targeted mutagenesis leads to simultaneous modification of different homoeologous gene copies in polyploid oilseed rape (Brassica napus). Plant Physiol. 174, 935–942. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brinkman, E.K. , Chen, T. , Amendola, M. and van Steensel, B. (2014) Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res. 42, e168–e168. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. De Buck, S. , Podevin, N. , Nolf, J. , Jacobs, A. and Depicker, A. (2009) The T‐DNA integration pattern in Arabidopsis transformants is highly determined by the transformed target cell. Plant J. 60, 134–145. [DOI] [PubMed] [Google Scholar]
  7. Caddick, S.E.K. , Harrison, C.J. , Stavridou, I. , Mitchell, J.L. , Hemmings, A.M. and Brearley, C.A. (2008) A Solanum tuberosum inositol phosphate kinase (StITPK1) displaying inositol phosphate–inositol phosphate and inositol phosphate–ADP phosphotransferase activities. FEBS Lett. 582, 1731–1737. [DOI] [PubMed] [Google Scholar]
  8. Campbell, L. , Rempel, C.B. and Wanasundara, J.P. (2016) Canola/rapeseed protein: future opportunities and directions‐workshop proceedings of IRC 2015. Plants (Basel) 5, 17. doi:10.3390/plants5020017 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chalhoub, B. , Denoeud, F. , Liu, S. , Parkin, I.A. , Tang, H. , Wang, X. , Chiquet, J. , Belcram, H. , Tong, C. , Samans, B. , Correa, M. , Da Silva, C. , Just, J. , Falentin, C. , Koh, C.S. , Le Clainche, I. , Bernard, M. , Bento, P. , Noel, B. , Labadie, K. , Alberti, A. , Charles, M. , Arnaud, D. , Guo, H. , Daviaud, C. , Alamery, S. , Jabbari, K. , Zhao, M. , Edger, P.P. , Chelaifa, H. , Tack, D. , Lassalle, G. , Mestiri, I. , Schnel, N. , Le Paslier, M.C. , Fan, G. , Renault, V. , Bayer, P.E. , Golicz, A.A. , Manoli, S. , Lee, T.H. , Thi, V.H. , Chalabi, S. , Hu, Q. , Fan, C. , Tollenaere, R. , Lu, Y. , Battail, C. , Shen, J. , Sidebottom, C.H. , Wang, X. , Canaguier, A. , Chauveau, A. , Berard, A. , Deniot, G. , Guan, M. , Liu, Z. , Sun, F. , Lim, Y.P. , Lyons, E. , Town, C.D. , Bancroft, I. , Wang, X. , Meng, J. , Ma, J. , Pires, J.C. , King, G.J. , Brunel, D. , Delourme, R. , Renard, M. , Aury, J.M. , Adams, K.L. , Batley, J. , Snowdon, R.J. , Tost, J. , Edwards, D. , Zhou, Y. , Hua, W. , Sharpe, A.G. , Paterson, A.H. , Guan, C. and Wincker, P. (2014) Plant genetics. Early allopolyploid evolution in the post‐Neolithic Brassica napus oilseed genome. Science (New York, NY) 345, 950–953. [DOI] [PubMed] [Google Scholar]
  10. Conley, D.J. , Paerl, H.W. , Howarth, R.W. , Boesch, D.F. , Seitzinger, S.P. , Havens, K.E. , Lancelot, C. et al. (2009) Controlling eutrophication: nitrogen and phosphorus. Science 323, 1014. [DOI] [PubMed] [Google Scholar]
  11. Cordell, D. , Drangert, J.‐O. and White, S. (2009) The story of phosphorus: Global food security and food for thought. Glob. Environ. Change 19, 292–305. [Google Scholar]
  12. Dersjant‐Li, Y. , Awati, A. , Schulze, H. and Partridge, G. (2015) Phytase in non‐ruminant animal nutrition: a critical review on phytase activities in the gastrointestinal tract and influencing factors. J. Sci. Food Agric. 95, 878–896. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dieck, C. , Boss, W. and Perera, I. (2012) A role for phosphoinositides in regulating plant nuclear functions. Front. Plant Sci. 3, 50. 10.3389/fpls.2012.00050. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Donahue, J.L. , Alford, S.R. , Torabinejad, J. , Kerwin, R.E. , Nourbakhsh, A. , Ray, W.K. , Hernick, M. et al. (2010) The Arabidopsis thaliana Myo‐Inositol 1‐Phosphate Synthase1 gene is required for Myo‐inositol synthesis and suppression of cell death. Plant Cell 22, 888. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Du, H. , Liu, L. , You, L. , Yang, M. , He, Y. , Li, X. and Xiong, L. (2011) Characterization of an inositol 1,3,4‐trisphosphate 5/6‐kinase gene that is essential for drought and salt stress responses in rice. Plant Mol. Biol. 77, 547–563. [DOI] [PubMed] [Google Scholar]
  16. El‐Batal, A.I. and Abdel, K.H. (2001) Phytase production and phytic acid reduction in rapeseed meal by Aspergillus niger during solid state fermentation. Food Res. Int. 34, 715–720. [Google Scholar]
  17. Fauser, F. , Schiml, S. and Puchta, H. (2014) Both CRISPR/Cas‐based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana . Plant J. Cell Mol. Biol. 79, 348–359. 10.1111/tpj.12554. [DOI] [PubMed] [Google Scholar]
  18. Gacek, K. , Bartkowiak‐Broda, I. and Batley, J. (2018) Genetic and molecular regulation of seed storage proteins (SSPs) to improve protein nutritional value of oilseed rape (Brassica napus L.) seeds. Front. Plant Sci. 9, 890–890. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gibson, R.S. , Raboy, V. and King, J.C. (2018) Implications of phytate in plant‐based foods for iron and zinc bioavailability, setting dietary requirements, and formulating programs and policies. Nutr. Rev. 76, 793–804. [DOI] [PubMed] [Google Scholar]
  20. Jasinski, S. , Chardon, F. , Nesi, N. , Lécureuil, A. and Guerche, P. (2018) Improving seed oil and protein content in Brassicaceae: some new genetic insights from Arabidopsis thaliana . OCL 25, D603. [Google Scholar]
  21. Jiang, M. , Liu, Y. , Liu, Y. , Tan, Y. , Huang, J. and Shu, Q. (2019) Mutation of Inositol 1,3,4‐trisphosphate 5/6‐kinase6 impairs plant growth and phytic acid synthesis in Rice. Plants 8, 114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Katoh, K. and Standley, D.M. (2013) MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–780. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kim, S.I. and Tai, T.H. (2011) Identification of genes necessary for wild‐type levels of seed phytic acid in Arabidopsis thaliana using a reverse genetics approach. Mol. Genet. Genomics 286, 119–133. [DOI] [PubMed] [Google Scholar]
  24. Kim, S.‐I. and Tai, T.H. (2014) Identification of novel rice low phytic acid mutations via TILLING by sequencing. Mol. Breed. 34, 1717–1729. [Google Scholar]
  25. Kumar, S. , Stecher, G. , Li, M. , Knyaz, C. and Tamura, K. (2018) MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evol. 35, 1547–1549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Letunic, I. and Bork, P. (2007) Interactive tree of Life (iTOL): an online tool for phylogenetic tree display and annotation. Bioinformatics 23, 127–128. [DOI] [PubMed] [Google Scholar]
  27. Letunic, I. and Bork, P. (2019) Interactive tree of Life (iTOL) v4: recent updates and new developments. Nucleic Acids Res. 47(W1), W256–W259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Liu, J. , Huang, S. , Sun, M. , Liu, S. , Liu, Y. , Wang, W. , Zhang, X. et al. (2012) An improved allele‐specific PCR primer design method for SNP marker analysis and its application. Plant Meth. 8, 34. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Loewus, F.A. and Murthy, P.P.N. (2000) myo‐Inositol metabolism in plants. Plant Sci. 150, 1–19. [Google Scholar]
  30. Matthäus, B. , Lösing, R. and Fiebig, H.‐J. (1995) Determination of inositol phosphates IP3 – IP6 in rapeseed and rapeseed meal by an HPLC method, Part 1: Method. Lipid/Fett 97, 289–291. [Google Scholar]
  31. Otegui, M.S. , Capp, R. and Staehelin, L.A. (2002) Developing seeds of Arabidopsis store different minerals in two types of vacuoles and in the endoplasmic reticulum. Plant Cell 14, 1311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Perera, I. , Seneweera, S. and Hirotsu, N. (2018) Manipulating the phytic acid content of rice grain toward improving micronutrient bioavailability. Rice (NY) 11, 4–4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Qin, Z.X. , Chen, Q.J. , Tong, Z. and Wang, X.C. (2005) The Arabidopsis inositol 1,3,4‐trisphosphate 5/6 kinase, AtItpk‐1, is involved in plant photomorphogenesis under red light conditions, possibly via interaction with COP9 signalosome. Plant Physiol. Biochem. 43, 947–954. [DOI] [PubMed] [Google Scholar]
  34. Raboy, V. (2000) Low‐phytic‐acid Grains. Food Nut. Bull. 21, 423–427. [Google Scholar]
  35. Raboy, V. (2003) myo‐Inositol‐1,2,3,4,5,6‐hexakisphosphate. Phytochemistry 64, 1033–1043. [DOI] [PubMed] [Google Scholar]
  36. Raboy, V. (2009) Approaches and challenges to engineering seed phytate and total phosphorus. Plant Sci. 177, 281–296. [Google Scholar]
  37. Raboy, V. , Gerbasi, P.F. , Young, K.A. , Stoneberg, S.D. , Pickett, S.G. , Bauman, A.T. , Murthy, P.P.N. et al. (2000) Origin and seed phenotype of maize low phytic acid 1–1 and low phytic acid 2–1 . Plant Physiol. 124, 355–368. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Reddy, M.B. , Hurrell, R.F. , Juillerat, M.A. and Cook, J.D. (1996) The influence of different protein sources on phytate inhibition of nonheme‐iron absorption in humans. Am. J. Clin. Nut. 63, 203–207. [DOI] [PubMed] [Google Scholar]
  39. Rounds, M.A. and Nielsen, S.S. (1993) Anion‐exchange high‐performance liquid chromatography with post‐column detection for the analysis of phytic acid and other inositol phosphates. J. Chromatogr. A 653, 148–152. [DOI] [PubMed] [Google Scholar]
  40. Sashidhar, N. , Harloff, H.‐J. and Jung, C. (2019) Identification of phytic acid mutants in oilseed rape (Brassica napus) by large scale screening of mutant populations through amplicon sequencing. New Phytol. 225, 2022–2034. [DOI] [PubMed] [Google Scholar]
  41. Shah, S. , Weinholdt, C. , Jedrusik, N. , Molina, C. , Zou, J. , Grosse, I. , Schiessl, S. , Jung, C. and Emrani, N. (2018) Whole‐transcriptome analysis reveals genetic factors underlying flowering time regulation in rapeseed (Brassica napus L.). Plant Cell Environ. 41, 1935–1947. [DOI] [PubMed] [Google Scholar]
  42. Shi, J. , Wang, H. , Wu, Y. , Hazebroek, J. , Meeley, R.B. and Ertl, D.S. (2003) The maize low‐phytic acid mutant lpa2 is caused by mutation in an inositol phosphate kinase gene. Plant Physiol. 131, 507. 10.1104/pp.014258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Shi, J. , Wang, H. , Schellin, K. , Li, B. , Faller, M. , Stoop, J.M. , Meeley, R.B. et al. (2007) Embryo‐specific silencing of a transporter reduces phytic acid content of maize and soybean seeds. Nat. Biotechnol. 25, 930–937. 10.1038/nbt1322. [DOI] [PubMed] [Google Scholar]
  44. Sparvoli, F. and Cominelli, E. (2015) Seed biofortification and phytic acid reduction: a conflict of interest for the plant? Plants (Basel) 4, 728–755. 10.3390/plants4040728. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Stiles, A.R. , Qian, X. , Shears, S.B. and Grabau, E.A. (2008) Metabolic and signaling properties of an Itpk gene family in Glycine max. FEBS Lett. 582, 1853–1858. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sweetman, D. , Stavridou, I. , Johnson, S. , Green, P. , Caddick, S.E.K. and Brearley, C.A. (2007) Arabidopsis thaliana inositol 1,3,4‐trisphosphate 5/6‐kinase 4 (AtITPK4) is an outlier to a family of ATP‐grasp fold proteins from Arabidopsis. FEBS Lett. 581, 4165–4171. [DOI] [PubMed] [Google Scholar]
  47. Tan, S.H. , Mailer, R.J. , Blanchard, C.L. and Agboola, S.O. (2011) Canola proteins for human consumption: extraction, profile, and functional properties. J. Food Sci. 76, R16–R28. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Tang, W. , Newton, R.J. and Weidner, D.A. (2006) Genetic transformation and gene silencing mediated by multiple copies of a transgene in eastern white pine. J. Exp. Bot. 58, 545–554. [DOI] [PubMed] [Google Scholar]
  49. Tang, Y. , Tan, S. and Xue, H. (2013) Arabidopsis inositol 1,3,4‐trisphosphate 5/6 kinase 2 is required for seed coat development. Acta Biochim. Biophys. Sin. 45, 549–560. [DOI] [PubMed] [Google Scholar]
  50. Thompson, L.U. (1990) Phytates in Canola/Rapeseed. In:Canola and rapeseed: production, chemistry, nutrition and processing technology ( Shahidi, F. ed.), pp. 173–192. Boston, MA: Springer, US. 10.1007/978-1-4615-3912-4_10. [DOI] [Google Scholar]
  51. Uppström, B. and Svensson, R. (1980) Determination of phytic acid in rapeseed meal. J. Sci. Food Agric. 31, 651–656. [Google Scholar]
  52. Wanasundara, J.P.D. , McIntosh, T.C. , Perera, S.P. , Withana‐Gamage, T.S. and Mitra, P. (2016) Canola/rapeseed protein‐functionality and nutrition. OCL 23, D407. [Google Scholar]
  53. Wang, Y. , Cheng, X. , Shan, Q. , Zhang, Y. , Liu, J. , Gao, C. and Qiu, J.‐L. (2014) Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nat. Biotechnol. 32, 947. [DOI] [PubMed] [Google Scholar]
  54. Yao, M.‐Z. , Zhang, Y.‐H. , Lu, W.‐L. , Hu, M.‐Q. , Wang, W. and Liang, A.‐H. (2012) Phytases: crystal structures, protein engineering and potential biotechnological applications. J. Appl. Microbiol. 112, 1–14. [DOI] [PubMed] [Google Scholar]
  55. Zarhloul, M.K. , Friedt, W. , Khoschkhoi‐Yazdi, M.R. and Liihs, W. (1999)Genetic transformation and shoot regeneration ability of resynthesised Brassica napus line ’RS 306’. EUCARPIA.
  56. Zhang, C. , Majerus, P.W. and Wilson, M.P. (2012) Regulation of inositol 1,3,4‐trisphosphate 5/6‐kinase (ITPK1) by reversible lysine acetylation. Proc. Natl. Acad. Sci. USA 109, 2290–2295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Zhang, Z. , Hua, L. , Gupta, A. , Tricoli, D. , Edwards, K.J. , Yang, B. and Li, W. (2019) Development of an Agrobacterium‐delivered CRISPR/Cas9 system for wheat genome editing. Plant Biotechnol. J. 17, 1623–1635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Zhao, J. , Jamar, D.C.L. , Lou, P. , Wang, Y. , Wu, J. , Wang, X. , Bonnema, G. et al. (2008) Quantitative trait loci analysis of phytate and phosphate concentrations in seeds and leaves of Brassica rapa . Plant Cell Environ. 31, 887–900. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1 Expression analysis of BnITPK paralogs in seeds and leaves.

Figure S2 Protein alignment of ITPK showing the inositol, ATP and Mg2+ binding sites.

Figure S3 Predicted protein structure of Bn.ITPK1.C09 in Haydn and in the mutant line 190666.

Table S1 Primer sequences used in this study.

Table S2 Editing status in regenerated T1 plants.

Table S3 Gene acronyms for four BnITPK genes used in this study.

Table S4 Segregation analysis in T3 generations.

PBI-18-2241-s001.pdf (1.3MB, pdf)

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