Plant Breeding Institute

Identification and characterization of mutations in the phytic acid biosynthesis pathway genes in oilseed rape

Background 

Oilseed rape (rapeseed, Brassica napus L., genome AACC, 2n=38) is the most important oilseed crop in Europe. It stands in second position for oil production in the world after soybean. Dry mature seeds from rapeseed accumulate oil (45-50%) and proteins (20-25%), while the meal remaining after oil extraction contains about 40% protein. The availability of rapeseed meal as a protein supplement in animal feed is limited due to the toxicity and digestibility problems caused by anti-nutritional components like sinapine, glucosinolates and phytate.

Phytic acid (m-inositol hexakisphosphate) is the principal storage form of phosphorus in B.napus seeds with levels ranging from 2-4% of the seed weight. Phytate provides 65 to 85% of the total phosphorus in seeds. Humans and non-ruminant animals lack phytase, the digestive enzyme to hydrolyze phytic acid, which reduces the amount of bio-available phosphorus in their diet. The negatively charged phytic acid molecule also chelates minerals such as calcium, potassium, magnesium, iron and zinc and lowers their bioavailability. In addition to the nutritional effects of phytate in animal diets, there are also environmental consequences as undigested phytic acid increases phosphate in animal manure and can pollute water resources.

Phytic acid has also other important functions in plants (DNA repair, signaling pathway, cell membrane formation, plant defense reactions etc.) and is synthesized via two pathways (lipid dependent and lipid independent). Many synthesis and transporter genes were identified previously in various crop species by mutant and knock-out analysis. This project aims at developing an efficient and high throughput method to knock out key genes involved in the phytic acid pathway in B.napus via Next Generation Sequencing of an EMS mutant population and a CRISPR-Cas9 approach to provide a deeper understanding of the biological function of various genes involved in the pathway.

Objectives

  • Identification and characterization of phytic acid synthesis and transporter genes in B. napus in silico and in planta
  • Detection of point mutations within B. napus phytic acid synthesis genes using TILLING by Illumina Next Generation Sequencing
  • Molecular and phenotypic characterization of single and double mutants
  • Knock-out of phytic acid synthesis genes by CRISPR-Cas9 genome editing
  • Expression profiling, enzyme and transporter measurements of selected target genes

Results

Eight biosynthetic genes and one transporter gene were identified in the phytic acid pathway for the production of phytic acid in Arabidopsis. The corresponding paralogs were selected in Brassica napus as target genes for knockout analysis. After expression analysis in developing seeds of control cultivar Express 617, we chose 23 paralogs of the 6 key synthesis and transporter genes BnITPK, BnMIK, BnIPK, Bn2-PGK2, BnIPK2ß and BnMRP5 for knock-out by CRISPR-Cas9 and 12 highly expressed paralogs of BnMIPS, BnIPK2ß, BnMIK, BnIPK, Bn2-PGK2,  and BnMRP5 for mutant screening by TILLING. The screening of our Express 617 EMS TILLING population resulted in successful identification of 1487 EMS mutations comprising 49 knock-out mutations. Combination of single mutants by crossing resulted in significant reductions in seed phytic acid contents in BnMRP5 double mutants (15%) and Bn2-PGK2 mutants (25%). The CRIPSR-Cas9 experiment resulted in an editing of three out of six targeted paralogs of BnITPK. The triple mutant showed a 30% reduction in seed phytic acid. Further crossing experiments are currently underway to combine the so-far characterized mutations in different genes to achieve further reduction in phytic acid.

Project team

Dr. Niharika Sashidhar

Dr. Hans-Joachim Harloff

Prof. Christian Jung

Scientific Partners

Prof. Dr. rer. nat. Wolfgang Bilger, Ökophysiologie der Pflanzen, University of Kiel

Institute of Clinical Molecular Biology (IKMB), University of Kiel

Commercial Partners

Norddeutsche Pflanzenzucht Hans-Georg Lembke KG

Publications

  • Sashidhar N, Harloff HJ, Jung C (2020) Knockout of MULTI-DRUG RESISTANT PROTEIN 5 genes lead to low phytic acid contents in oilseed rape Frontiers in Plant Science, Frontiers in Plant Science 11 (603). doi:10.3389/fpls.2020.00603
  • Sashidhar N, Harloff HJ, Potgieter L, Jung C (2020) Gene editing of three BnITPK genes in tetraploid oilseed rape leads to significant reduction of phytic acid in seeds. Plant Biotechnology Journal in press, doi: 10.1111/pbi.13380
  • Sashidhar N, Harloff H, Jung C (2019) Identification of phytic acid mutants in oilseed rape (Brassica napus L.) by large scale screening of mutant populations through amplicon sequencing. New Phytologist 225:2022–2034

Posters and Oral Presentations

  • Sashidhar, N. (2019). Discovering novel phytic acid mutants in oilseed rape for future breeding. 15th International Rapeseed conference June 16-19, 2019, BCC Berlin. Oral Presentation.
  • Sashidhar, N. (2019). Breeding towards low phytic acid content in Brassica napus, 2019, Genome Research for Plant Breeding, University of Hohenheim, Stuttgart.-28.03.2019. Oral Presentation.
  • Sashidhar, N. (2019) ., Harloff, H.-J., Jung, C. High throughput reverse genetic tools for knocking out several genes of the phytic acid pathway in Brassica napus, International Plant & Animal Genome XXVII, San Diego, 12.-16.01.2019- Poster Presentation.
  • Sashidhar, N. (2018). High throughput reverse genetic tools for knocking out several genes of the phytic acid pathway in Brassica napus, Young Scientists Meeting 2018, JKI, Braunschweig.-14.11.2018. Oral Presentation.
  • Sashidhar, N. (2018) Genome editing strategies for multiple knock out of phytic acid pathway gene families in Brassica napus, PGSC 2018, IPK, Gaterleben .-06.06.2018. Oral Presentation.
  • Sashidhar, N. (2018). Reverse genetic tools for mutational analyses of all genes of a complex metabolic pathway in B. napus. Kiel Plant Science symposium 2018, Kiel.-31.05.2018. Oral Presentation.
  • Harloff.H.,  Sashidhar, N., Blanc,Noelle., Jung,C.  (2017): Systematic knock-out study of phytic acid synthesis genes in Brassica napus, Botanikertagung 2017, Kiel, 17.-21.09.2017. Poster Presentation.
  • Harloff.H., Sashidhar, N., Jung,C.  (2017): CRISPR/Cas9 targeted mutagenesis in phytic acid synthesis genes of Brassica napus PP1530 International Workshop: ‘Genome Engineering’, Gatersleben, 22-23.06.2017. Poster Presentation.
  • Harloff.H.,  Sashidhar, N., Jung,C.   (2018): Knock-out study of phytic acid synthesis genes in Brassica napus, German Plant Breeding conference-Wernigerode, 28.02.2018.Poster Presentation.
  • Harloff.H., (2016): Use of TILLING and Genome Editing to Reduce Phytic Acid in Rapeseed, Sino German Conference-Kiel, 25.05.2016. Oral Presentation.

Financial Support (past three years)

Funding has been provided by the DFG grant No. JU 205/26-1

Updated: 16.07.2020

Responsible for this webpage: Dr. H.-J. Harloff