GERMACRENE A SYNTHASE MUTANTS

Abstract

The invention is in the field of agriculture, in particular in the field of crop improvement for processing, more particularly in the field of sesquiterpene lactone (STL), squalene and phenolic compound biosynthesis by plants. A method for producing a plant having reduced STL levels, increased squalene levels and increased phenolic compound levels is disclosed, as well as a plant produced by such method.

Claims

1. A method for producing a plant having, compared to a control plant, at least one of: (a) reduced sesquiterpene lactone (STL) level; (b) increased squalene level; and (c) increased level of a phenolic compound, the method comprising mutating one or more endogenous functional germacrene A synthase (GAS)-short genes in the plant, resulting in a decreased or abolished expression or activity of one or more functional GAS-short proteins.

2. The method according to claim 1, wherein the one or more GAS-short genes encode a protein having at least 70% sequence identity with any one of SEQ ID NO: 1-6.

3. The method according to claim 1, wherein multiple, preferably all, endogenous functional GAS-short genes in the plant are mutated.

4. The method according to claim 3, wherein all endogenous functional GAS-short genes in the plant are mutated.

5. The method according to claim 1, wherein at least one nucleotide is inserted, deleted or substituted in a coding sequence of the one or more GAS-short genes, resulting in decreased or abolished activity of an encoded GAS-short proteins.

6. The method according to claim 1, wherein at least one nucleotide is inserted, deleted or substituted in at least one transcription regulatory sequence of the one or more GAS-short genes, resulting in decreased or abolished expression of an encoded GAS-short proteins.

7. The method according to claim 1, wherein the one or more endogenous functional GAS-short genes are any one of CiGAS-S1, CiGAS-S2 and CiGAS-S3, or a homologue thereof.

8. The method according to claim 1, wherein the expression of the protein is impaired in at least any one of the leaves and the roots of the plant.

9. The method according to claim 1, further comprising regenerating the plant by at least one of: (a) inulin extraction; (b) squalene extraction; and (c) phenolic compound extraction, from the plant.

10. The method according to claim 9, comprising regenerating the plant by at least one of: (a) inulin extraction; (b) squalene extraction; and (c) phenolic compound extraction, from the plant root.

11. A nucleic acid comprising a germacrene A synthase (GAS)-short gene having one or more modifications, resulting in impaired expression or activity of a functional GAS-short protein when the nucleic acid is present in a plant as compared to an identical nucleic acid not having the one or more modifications.

12. The nucleic acid according to claim 10, wherein the functional GAS-short protein has at least 70% sequence identity with any one of SEQ ID NO: 1-6.

13. A construct, vector or host cell comprising the nucleic acid of claim 9.

14. A plant obtainable from a method according to claim 1, or progeny thereof.

15. A plant having at least one of: (a) a reduced sesquiterpene lactone (STL) level; (b) an increased squalene level; and (c) an increased level of a phenolic compound, as compared to a control plant, wherein the plant exhibits reduced expression and/or reduced activity of a functional germacrene A synthase (GAS)-short protein, or progeny thereof.

16. The plant according to claim 15, comprising a mutation in one or more endogenous functional GAS-short genes.

17. The plant according to claim 16, comprising a mutation in all of the endogenous functional GAS-short genes.

18. The plant according to claim 15, wherein the functional GAS-short protein has at least 70% sequence identity with any one of SEQ ID NO: 1-6.

19. The plant according to claim 15, comprising a nucleic acid having a germacrene A synthase (GAS)-short gene having one or more modifications.

20. A method of producing at least one of inulin, squalene and a phenolic compound, the method comprising: (a) providing a plant having at least one of: (i) reduced sesquiterpene lactone (STL) level; (ii) increased squalene level; and (iii) increased level of a phenolic compound, as compared to a control plant, wherein the plant exhibits reduced expression and/or reduced activity of a functional germacrene A synthase (GAS)-short protein, or progeny thereof; (b) extracting at least one of inulin, squalene and a phenolic compound from said plant or plant part; and (c) optionally, purifying at least one of said inulin, squalene and a phenolic compound.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0174] FIG. 1: Alignment of exon 4 sequences of Cichorium intybus GAS genes and indication of the sequence targeted by the guide RNAs. The underlines indicate the target sequences of the guide RNAs (the sequence of GAS-S1, GAS-S2, GAS-S3 and GAS-L correspond to respectively SEQ ID NOs: 67, 68, 69 and 70).

[0175] FIG. 2: Indel mutations of the five selected mutant lines (MT1 to MT5) are shown. For each gene the target site is shown underlined with the mutations present in each allele shown underneath each target. Alleles without indels are indicated as wild type.

[0176] FIG. 3: STL (lactucin, lactucopicrin and 8-deoxylactucin) levels expressed in leaves of the different mutant (MD and control lines (WT). The genotypes for each line is provided in the table underneath the x-axis, wherein a “+” means a wild type allele, and a “−” means a mutated allele. Specific mutations are provided in FIG. 2.

[0177] FIG. 4: STL (lactucin 15-oxalate, lactucopicrin 15-oxalate and 8-deoxylactucin 15-oxalate) levels in leaves of the different mutant control lines. The genotypes for each line is provided in the table underneath the x-axis, wherein “+” means a wild type allele, and “−” means a mutated allele. Specific mutations are provided in FIG. 2.

[0178] FIG. 5: STL (lactucin, lactucopicrin and 8-deoxylactucin) levels in roots of the different mutant (MT) and control lines NOT The genotypes for each line is provided in the table underneath the x-axis, wherein a “+” means a wild type allele, and a “−” means a mutated allele. Specific mutations are provided in FIG. 2.

[0179] FIG. 6: STL (lactucin 15-oxalate, lactucopicrin 15-oxalate and 8-deoxylactucin 15-oxalate) levels in roots of the different mutant control lines. The genotypes for each line is provided in the table underneath the x-axis, wherein “+” means a wild type allele, and “−” means a mutated allele. Specific mutations are provided in FIG. 2.

[0180] FIG. 7: GC-MS chromatogram of chicory root tissues. Peak1-3: acetylated triterpenes; Peak 4: squalene; Peak 5: stigmasterol; Peak 6: sitosterol.

[0181] FIG. 8: Increase of phenolic compounds in leaves and roots of chicory GAS KO lines.

EXAMPLE 1

[0182] In this example we describe the isolation of protoplasts from chicory leaves which were then transfected with different CRISPR/Cas9 reagents targeting the GAS genes, in order to also investigate efficiency of these different reagents and feasibility of the technology to induce indels in multiple genes and alleles at the same time in chicory. These protoplasts were then regenerated into mature plants with mutations in different GAS genes which were then phenotyped for STL content. Surprisingly, we found that inactivation of the GAS-short genes without affecting the GAS-long gene, almost fully blocked STL production in both leaf and root tissue.

Isolation of Chicory Protoplasts

[0183] Protoplast isolation, transfection and culture was performed as previously described (Frearson et al. Dev Biol, 1973, 33, 130-137; Kao et al. Planta, 1975, 126, 105-110; Negrutiu et al. Plant. Mol. Biol. 1978, 8, 363-373; Nenz et al. Plant Cell Tiss Org, 2000, 62, 85-88; Deryckere et al. Plant Cell Rep 2012, 31, 2261-2269) with several modifications. In vitro shoot cultures of Cichorium intybus var. sativa (Orchies C37) were maintained on MS20 medium with 0.8% agar in high plastic jars at 16/8 h photoperiod of 100 μmol.Math.m.sup.−2.Math.s.sup.−1 PPF at 25° C. and 60-70% RH. Young leaves (10-12) were harvested, placed in a dish containing 5 ml CPW9M medium (Frearson et al. Dev Biol, 1973, 33, 130-137) and were gently sliced perpendicularly to the mid nerve to ease the penetration of the enzyme mixture. Sliced leaves were transferred to a dish containing 25 ml CPW9M and an enzyme mixture (1% (w/v) Cellulase Onozuka RS, 0.2% (w/v) Macerozyme Onozuka R10). Digestion was carried out at 25° C. for 14-16 h, in the dark. The protoplasts were filtered through a 50 μm stainless steel sieve and were harvested by centrifugation for 5 minutes at 85×g. Protoplasts were resuspended in 1 ml CPW9M medium and then added to a tube containing 5 ml CPW13S-1.2M. This was then centrifuged for 10 minutes at 85×g at RT. Live protoplasts were then harvested from the interface layer, transferred to a fresh tube and then mixed with 11 ml CPW9M. The protoplast density was then determined in a haemocytometer.

Transfection and CRISPR/Cas9 Mutagenesis of Chicory GAS Genes

[0184] Exon 4 of the GAS-family enzymes encodes a region of the protein that makes up part of the GAS active site. Chicory leaf protoplasts were transfected either with CRISPR-Cas9/guide RNA complexes (RNPs) or plasmids encoding the same using guide RNAs targeting exon 4 in the GAS-short and GAS-long genes (i.e. targeting SEQ ID NO: 22, 23 and 24, respectively; see also FIG. 1). RNPs were made by combining 10 μg SpCas9-NLS protein (New England Biolabs) and 10 μg of a guide RNA in 1×SpCas9 reaction buffer (New England Biolabs) in a final volume of 20 μl. For plasmid based transfection, a plasmid encoding the guide RNAs operably linked to an Arabidopsis U6 promoter and a plasmid carrying the SpCas9 ORF operably linked to an Arabidopsis ubiquitin promoter promoter were mixed at a 1:3 molar ration. For each transfection the reagents, i.e. 20 μg RNPs or 80 μg plasmids encoding the same, were mixed with 0.25×10.sup.6 protoplasts in a total volume of 250 μl MaMg medium and 250 μL PEG solution (400 g/l poly(ethylene glycol) 4000, Sigma-Aldrich #81240; 0.1 M Ca(NO.sub.3).sub.2) was then added. The transfection was then allowed to take place for 20 minutes at room temperature followed by the addition of 5 ml 0.275 M Ca(NO.sub.3).sub.2 solution which was thoroughly, but gently mixed in. The protoplasts were harvested by centrifugation for 5 minutes at 85×g and resuspended in 0.25 ml 9M culture medium.

Generation of GAS Mutant Plants

[0185] Transfected protoplasts were centrifuged at 85×g for 5 minutes at RT and then resuspended at a density of 0.10×10.sup.5 cells/ml in 5 ml 9M medium. An equal volume of alginate solution was then added dropwise and mixed thoroughly, and 1 ml of the mixture was then layered on a Ca-Agar plate (5 cm dish), dispersing the mixture evenly over the whole plate surface to form a disc. The alginate was allowed to polymerize for one hour and was then transferred to a 5 ml culture dish containing 4 ml K1Cg medium. After 7 days of culture in the dark at 28° C. the liquid culture medium was replaced with 4 ml K5CgK medium and the discs were cultured for a further 7 days using the same conditions. The discs were then cut into 5 mm broad strips and transferred to 9 cm plates with B5g-10-0, 2-SP-NB medium, two discs per plate. These were then incubated at 25° C. in the dark for two to three weeks whereupon the microcalli formed were then picked with tweezers and transferred to MS10-IB plates and incubated at 25° C. under low light for the first week followed by full light for the reminder of the regeneration. Calli were transferred to fresh MS10-IB medium every 3-4 weeks until signs of regeneration appeared. The developing shootlets were harvested and rooted on MS20 medium. Regenerated plants were then genotyped for mutations in the different GAS genes.

Genotyping Chicory Plants

[0186] Genomic DNA was isolated from regenerated chicory plants using the Maxwell Plant DNA kit (Promega) and the target sites in each gene were then amplified separately using specific forward primers (SEQ ID NO: 45 for GAS-S1, SEQ ID NO: 46 for GAS-S2, SEQ ID NO: 47 for GAS-S3 and SEQ ID NO: 48 for GAS-L1) and reverse primers (SEQ ID NO: 49 for GAS-S1, SEQ ID NO: 50 for GAS-S2, SEQ ID NO: 51 for GAS-S3 and SEQ ID NO: 52 for GAS-L1) primers. A nested PCR was then done on each PCR product using the appropriate forward primers (SEQ ID NO: 53 for GAS-S1 and GAS-S2, SEQ ID NO: 54 for GAS-S3 and SEQ ID NO: 55 for GAS-L1) and reverse primers (SEQ ID NO: 56 for GAS-S1 and GAS-S2, SEQ ID NO: 57 for GAS-S3 and SEQ ID NO: 58 for GAS-L1) and a final third PCR was then done with barcoded Illumina primers to enable later identification of the sequences. All of the these PCR products were then pooled and paired-end sequenced on an Illumina MiSeq apparatus. The sequences were then analyzed for the presence of indel mutations at the target sites.

[0187] Three mutant lines were selected, MT1, MT2, MT3, MT4 and MT5. As indicated in more detail in FIG. 2, MT1 comprises mutations in all alleles of all four GAS genes; MT2 comprises mutations in all GAS alleles except for the GAS-S2 alleles, which has the wild type sequence; MT3 comprises mutations in all GAS-short alleles, while the two GAS-L1 alleles do not comprise a mutation; MT4 comprises mutations in both GAS-S1 and GAS-S2 alleles and in one GAS-S3 allele, while the GAS-L1 alleles and one GAS-S3 allele did not comprise a mutation; and M5 comprises mutations only in both GAS-S1 alleles and one GAS-S3 allele, while the other GAS alleles do not comprise mutations.

[0188] The selected lines were transferred to the greenhouse for further phenotypic analysis. As controls, lines were also selected, which had also been regenerated from protoplasts but lacked any mutations in the GAS genes.

Quantification of Sesquiterpene Lactone Guaianolides

[0189] Sesquiterpene lactone content was determined in the leaves and roots of the five GAS mutant lines and the control plants. Chicory leaf and root material (100 mg) was frozen and powdered in liquid nitrogen. Extraction was performed using 77% methanol containing formic acid (0.1%), the samples were then vortexed, sonicated for 15 min and then centrifuged at 21000 g at room temperature.

[0190] The clear supernatant was transferred to a fresh vial and used for LC-MS analysis. LC-MS analysis was performed using the LC-PDA-LTQ-Orbitrap FTMS system (Thermo Scientific) which consist of an Acquity UPLC (H-Class) with Acquity elambda photodiode array detector (220-600 nm) connected to a LTQ/Orbitrap XL hybrid mass spectrometer equipped with an electrospray ionizator (ESI). The injection volume was 5 μl. Chromatographic separation was on a reversed phase column (Luna C18/2, 3μ, 2.0×150 mm; Phenomenex, USA) at 40° C. Degassed eluent A [ultra-pure water: formic acid (1000:1, v/v)] and eluent B [acetonitrile:formic acid (1000:1, v/v)] were used at a flow rate of 0.19 ml min-1. A linear gradient from 5 to 75% acetonitrile (v/v) in 45 min was applied, which was followed by 15 min of washing and equilibration. FTMS full scans (m/z 90.00-1350.00) were recorded with a resolution of 60,000.

[0191] The samples were analyzed for the presence of six STLs (lactucin, lactucin-15-oxalate, 8-deoxylactucin, 8-deoxylactucin 15-oxalate, lactucopicrin and lactucopicrin 15-oxalate). The levels of these compounds in the leaves of the mutant and control plants are shown in FIGS. 3 and 4. The levels of these compounds in the root of the mutant and control plants are shown in FIGS. 5 and 6. The total peak area of each compound was quantified.

Results

[0192] The level of STLs in the two control lines was broadly similar, showing that the regeneration process had not introduced a large amount of STL variation. However, several lines containing mutations in the GAS genes showed a strong reduction in the amount of STLs produced in the leaves and roots. There appears to be a direct correlation between the type of functional GAS genes present and the levels of STLs produced. MT1, containing mutations in all of the GAS genes, shows the lowest STL levels, while the next highest expresser (MT3), lacks functional copies of the GAS-S1/S2/S3 genes but retains the GAS-L1 gene. M4, only lacking the GAS-S1/S2 genes and one GAS-S3 allele, shows reduced STL production by approximately 70%. These results demonstrate that the GAS-S1 and GAS-S2 genes seem to be responsible for most of the STL production in the leaves and roots, with the lines lacking both of these genes (MT1, MT3 and MT4) showing the largest decreased STL levels. MT2, having two functional GAS-S2 alleles still produces approximately 75% of the wild type levels, while MT1 that lacks any functional GAS gene, production is almost eliminated, suggesting that GAS-S2 is most important for sesquiterpene lactone production in the leaves and root. The activity of GAS-L1 seems to be low, as shown by the difference between the MT3 only having retained the GAS-L1 gene and MT1 lacking functional copies of all GAS genes.

[0193] Surprisingly, these results are in contrast to the state of the art that suggests GAS-long to be the most relevant GAS gene for STL accumulation. This study shows that inactivation of only one or two GAS-short genes significantly reduces the production of all the STLs assayed to approximately the same extent. Inactivation of all the GAS-short genes nearly abolished STL production in both leaf and root tissue. Several studies have shown that the GAS-long gene is predominantly expressed in leaves, but surprisingly this data demonstrates that mutations in the GAS-short genes have a greater effect on STL accumulation in the leaves and root than mutations in the GAS-long gene and therefore should be targeted to decrease STL levels in chicory.

EXAMPLE 2

Squalene Accumulation in Chicory GAS KO Lines

[0194] Chicory root and leaf material (300 mg) from 2 WT chicory plants (WT1 and WT2; see Example 1) and 5 edited chicory plants (MT1, MT2, MT3, MT4, MT5; see Example 1) carrying a deletion of the GAS synthase gene was analyzed. Plant material was frozen and powdered in liquid N.sub.2. The samples were then extracted with 1.5 ml of hexane:ethyl acetate mixture (v/v 85:15). Samples were sonicated for 15 min in a sonication bath and centrifuged for 10 min at 1200 rpm. The extracts were dried over a Na.sub.2SO.sub.4 column prepared in a glass wool plugged glass pipette. Analytes from 1 μL samples were separated using a gas chromatograph (5890 series II, Hewlett-Packard) equipped with a 30 m×0.25 mm, 0.25 mm film thickness column (ZB-5, Phenomenex) using helium as carrier gas at flow rate of 1 ml/min. The injector was used in splitless mode with the inlet temperature set to 250° C. The initial oven temperature of 45° C. was increased after 1 min to 310° C. at a rate of 10° C./min and held for 5 min at 300° C. The GC was coupled to a mass-selective detector (model 5972A, Hewlett-Packard), scanning from 45 to 500 atomic mass units. Experimental samples were compared with authentic standards of squalene (Sigma-Aldrich), campesterol (Sigma-Aldrich), stigmasterol (Extrasynthese) and sitosterol (Extrasynthese) for verification.

[0195] In the semi-polar methanolic extract of chicory roots the effect of GAS deletion on accumulation of sesquiterpene lactones was studied by LC-MS, as described in Example 1.

[0196] The hexane extract of chicory root was examined for accumulation of terpenes and sterols by GC-MS. Other than trace amounts of farnesene and farnesol in the chicory lines having the highest reduction of STLs (MT1, MT3 and MT4), no accumulation of monoterpenes and sesquiterpenes was observed as compared to WT lines. However, a large new peak was detected in the chromatogram of these lines at the retention time of 26.7 min (see FIG. 7). This compound was identified as squalene by comparison of the mass spectrum to the NIST mass spectral library. The identification was verified by comparison of the retention time and mass spectrum to the authentic standard of squalene. The amount of squalene accumulating in the root was quantified at 154 ug/gFW, 99 ug/gFW and 55 ug/gFW in chicory lines MT3, MT1 and MT4, respectively. No squalene peak was observed in chicory root extracts of lines MT2 and MT5 nor in the extract of the wild-type chicory plants. Therefore, it seems that farnesyl pyrophosphate (FPP, C15) in the chicory roots that would normally be converted to germacrene A by activity of GAS enzymes became available and was converted by the activity of endogenous chicory squalene synthase to squalene (C30).

[0197] Squalene is a precursor for the biosynthesis of triterpenes and phytosterols. Wild-type chicory roots accumulate small amounts of acetylated-triterpenes (peak 1-3, elemental formula C32H5202, MW=468; see FIG. 7). Upon comparison of the wild-type plants to the GAS KO lines no increase in the amount of triterpenes was observed in any of the KO lines. The accumulation of phytosterols sitosterol, campesterol and stigmasterol in GAS KO lines was next compared to the WT chicory plants. Sitosterol was the major observed sterol in the root tissue of WT chicory plants (see FIG. 7). In lines MT3 and MT4 2.3-fold and 1.7-fold increase in the level of sitosterol was observed compared to the WT lines, yielding 42 ug/g FW and 32 ug/g FW sitosterol, respectively. WT levels of sitosterol were observed for line MT1, MT2 and MT5. The amount of stigmasterol and campestrol was below 5 ug/g FW for both WT and KO lines and therefore close to the detection limit of the GC-MS method and was not quantified (see FIG. 7).

[0198] The GC-MS analysis of the chicory leaves revealed that squalene accumulated to a much lesser extend in the leaves of chicory. In line MT1 only a very minor accumulation of squalene was detected at 13 ug/g FW and the other KO lines did not show increased squalene accumulation in the leaves. No additional accumulation of monoterpenes, sesquiterpenes, triterpenes or sterols beyond WT levels was observed in the leaves of chicory GAS KO lines.

EXAMPLE 3

Increase of Phenolic Compounds in Chicory GAS KO Lines

[0199] Chicory leaf and root material (100 mg) of the WT1, WT2, MT1, MT2, MT3, MT4, MT5 plants (see Example 1) was frozen and powdered in liquid N.sub.2. Extraction was performed using 77% methanol containing formic acid (0.1%), the samples were then vortexed, sonicated for 15 min and centrifuged at 21000 g at room temperature. The clear supernatant was transferred to a fresh vial and used for LC-MS analysis. LC-MS analysis was performed using the LC-PDA-LTQ-Orbitrap FTMS system (Thermo Scientific) which consist of an Acquity UPLC (H-Class) with Acquity elambda photodiode array detector (220-600 nm) connected to a LTQ/Orbitrap XL hybrid mass spectrometer equipped with an electrospray ionizator (ESI). The injection volume was 5 μl. Chromatographic separation was on a reversed phase column (Luna C18/2, 3˜, 2.0×150 mm; Phenomenex, USA) at 40° C. Degassed eluent A [ultra-pure water: formic acid (1000:1, v/v)] and eluent B [acetonitrile:formic acid (1000:1, v/v)] were used at a flow rate of 0.19 ml min-1. A linear gradient from 5 to 75% acetonitrile (v/v) in 45 min was applied, which was followed by 15 min of washing and equilibration. FTMS full scans (m/z 90.00-1350.00) were recorded with a resolution of 60,000.

[0200] The PDA spectrum of the samples was examined at the wavelength of 320 nm for detection of phenolic compounds. In the chicory root tissues 3,5-dicaffeoylquinic acid (elemental formula C25H24O12, [M+H].sup.+=517,13405) and chlorogenic acid (elemental formula C16H1809, [M+H].sup.+=355,10235) were observed as major phenolic compounds. In chicory leaves the major accumulated phenolic compounds observed were chlorogenic acid and chicoric acid (Peak 3, C22H18012, [M+H].sup.+=475.08710). The compounds were identified by accurate mass determination and comparison with authentic standards of chicoric acid, chlorogenic acid and 3,5-dicaffeoylquinic acid (Sigma-Aldrich). Surprisingly, an increase of phenolic compounds was observed in the chicory KO lines (see FIG. 8). The phenolic and terpene biosynthetic pathways are not directly related and do not source from the same pool of precursor and intermediates therefore the increase of phenolic compounds upon deletion of the GAS gene is unexpected. Chlorogenic acid accumulation was increased 3.8-fold, 3.0-fold and 1.7-fold in the roots of chicory KO lines MT1, MT3 and MT4, respectively. 3,5-dicaffeoylquinic acid was increased 5.6-fold, 4.0-fold and 1.9-fold in the roots of lines MT1, MT3 and MT4, respectively. Wild-type levels of chlorogenic acid and 3,5-dicaffeoylquinic acid were observed in roots of MT2 and MT5 lines. In the leaves increase of phenolic compounds was less pronounced. Increased level of chlorogenic acid was observed in lines MT1, MT2, MT3 up to maximally 2.6-fold in MT3. The content of chicoric acid was similarly increased in the leaves of the chicory KO lines MT1, MT2, MT3.