Simultaneous Targeting of Multiple Gene Homeologs to Alter Seed Oil Production in Camelina sativa

Abstract

The ability to transform Camelina sativa easily with biosynthetic enzymes derived from other plants has made this oil seed crop an ideal platform for the production of unusual lipids valuable for different applications. However, in addition to expressing transgenic enzymes, the suppression of endogenous enzyme activity to reduce competition for common substrates or cofactors is also required to enhance the production of target compounds. As camelina possesses a relatively undifferentiated hexaploid genome, up to three gene homeologs can code for any particular enzymatic activity, complicating efforts to alter endogenous biosynthetic pathways. New genome editing technologies, such as that offered by the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) system, offer the capability to introduce mutations into specifically targeted genomic sites. Here, by using a carefully designed guide RNA identical to all three homeologs, we demonstrate the ability of the CRISPR/Cas genome editing system to introduce mutations in all three CsDGAT1 or CsPDAT1 homeologous genes important for triacylglycerol (TAG) synthesis in developing seeds. Sequence analysis from transgenic T1 plants revealed that each CsDGAT1 or each CsPDAT1 homeolog was altered by multiple mutations, resulting in a genetic mosaic in the plants. Interestingly, seed harvested from both CsDGAT1- and CsPDAT1-targeted lines was often shrunken and wrinkled. Further, lipid analysis revealed that many lines produced seed with reduced oil content and altered fatty acid composition, consistent with the role of the targeted genes in seed oil biosynthesis. The CRISPR/Cas system therefore represents a useful method to alter endogenous biosynthetic pathways efficiently in polyploid species such as camelina.

Properties and Biotechnological Applications of Acyl‐CoA:diacylglycerol Acyltransferase and Phospholipid:diacylglycerol Acyltransferase from Terrestrial Plants and Microalgae

“…TAG can also be synthesized through acyl‐CoA‐independent pathways via the catalytic action of PDAT, which catalyzes the transfer of an acyl moiety from the sn ‐2 position of phosphatidylcholine (PtdCho) to the sn ‐3 position of sn ‐1, 2‐DAG to yield TAG (Dahlqvist et al, ; Ståhl et al, ). Both DGAT and PDAT play crucial roles in determining the flux of carbon into TAG (Aznar‐Moreno and Durrett, ; Harwood et al, ; Zhang et al, ). They also contribute to the routing of modified fatty acids from PtdCho into TAG in some plant species, such as flax ( Linum usitatissimum ), castor ( Ricinus communis ), tung tree ( Vernicia fordii ), and ironweed ( Vernonia galamensis ), which produce relatively high levels of polyunsaturated or unusual fatty acids in their seed oils (Kim et al, ; Kroon et al, ; Li et al, ; Pan et al, ; Shockey et al, ; van Erp et al, ).…”

“…Considering medium chain fatty acids such as 12:0 are rarely incorporated into the sn ‐2 of PtdCho, which is the substrate of PDAT, it is likely that the relative use of PDAT and DGAT in TAG biosynthesis in leaves is dependent on the substrates and acyl flux conditions within the cell (Bates, ). Besides Arabidopsis, PDAT genes have also been identified and characterized in various plant and microalgal species, including castor (Kim et al, ; van Erp et al, ), flax (Pan et al, ), Camelina sativa (Aznar‐Moreno and Durrett, ; Yuan et al, ), green algae C. reinhardtii (Yoon et al, ), and green algae Myrmecia incise (Liu et al, ). It should be noted that the PDAT nomenclature in the literature lacks consistency.…”

“…Although a T‐DNA insertion in the AtPDAT1 locus led to no effect on the fatty‐acid content or composition in Arabidopsis (Mhaske et al, ), Aznar‐Moreno and Durrett () introduced mutations in genes encoding PDAT1 in C. sativa (an AtPDAT1 homolog) using the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR‐associated protein) system and observed reduced seed oil content and altered fatty‐acid composition (e.g. decreased linoleic acid content) in many transgenic lines, supporting the contribution of PDAT1 in seed oil biosynthesis.…”

Nonspecific phospholipase C6 increases seed oil production in oilseed Brassicaceae plants

“…In addition, the reverse reaction of CDP‐choline: DAG cholinephosphotransferase (CPT), which converts PC to DAG, was speculated to be involved in TAG synthesis (Slack et al , 1983), but the role of this reverse reaction in TAG content and fatty acid composition is not documented. PDAT can transfer fatty acid directly from PC to DAG to form TAG (Dahlqvist et al , 2000), and knockout of PDAT in Camelina decreased seed oil content and changed fatty acid composition (Aznar‐Moreno & Durrett, 2017). In Arabidopsis, however, knockout of PDAT did not alter seed oil content (Mhaske et al , 2005) except when its expression was suppressed in the DGAT1 ‐null background (Zhang et al , 2009).…”

Use of CRISPR systems in plant genome editing: toward new opportunities in agriculture

“…To study the agricultural and industrial applications of CRISPR/Cas systems in plants, 52 articles dealing with trait improvement of crops were selected to assess how scientists are directing their use. The use of CRISPR/Cas systems covers various applications, from biotic stress tolerance to abiotic stress tolerance, and also includes the achievements of improved yield performance, biofortification and enhancement of plant quality (Table 1 and Figure 1).…”