WRINKLED1 Rescues Feedback Inhibition of Fatty Acid Synthesis in Hydroxylase-Expressing Seeds

Abstract

Previous attempts at engineering Arabidopsis (Arabidopsis thaliana) to produce seed oils containing hydroxy fatty acids (HFA) have resulted in low yields of HFA compared with the native castor (Ricinus communis) plant and caused undesirable effects, including reduced total oil content. Recent studies have led to an understanding of problems involved in the accumulation of HFA in oils of transgenic plants, which include metabolic bottlenecks and a decrease in the rate of fatty acid synthesis. Focusing on engineering the triacylglycerol assembly mechanisms led to modest increases in the HFA content of seed oil, but much room for improvement still remains. We hypothesized that engineering fatty acid synthesis in the plastids to increase flux would facilitate enhanced total incorporation of fatty acids, including HFA, into seed oil. The transcription factor WRINKLED1 (WRI1) positively regulates the expression of genes involved in fatty acid synthesis and controls seed oil levels. We overexpressed Arabidopsis WRI1 in seeds of a transgenic line expressing the castor fatty acid hydroxylase. The proportion of HFA in the oil, the total HFA per seed, and the total oil content of seeds increased to an average of 20.9%, 1.26 µg, and 32.2%, respectively, across five independent lines, compared with 17.6%, 0.83 µg, and 27.9%, respectively, for isogenic segregants. WRI1 and WRI1-regulated genes involved in fatty acid synthesis were up-regulated, providing for a corresponding increase in the rate of fatty acid synthesis.

Characterization and Ectopic Expression of CoWRI1, an AP2/EREBP Domain-Containing Transcription Factor from Coconut (Cocos nucifera L.) Endosperm, Changes the Seeds Oil Content in Transgenic Arabidopsis thaliana and Rice (Oryza sativa L.)

“…Over-expression of ZmWRI1 increases the fatty acid content of the mature maize grain, the contents of certain amino acids, several compounds involved in amino acid biosynthesis, and two intermediates of the tricarboxylic acid cycle (Pouvreau et al, 2011). Similar attempts were also carried out in A. thaliana and resulted in a 10–40% increase in seed oil content, and increased seed size and mass (Adhikari et al, 2016; Kanai et al, 2016). Although WRI1 is the best understood example of the transcription factors that have been identified in A. thaliana seeds, only a few have been characterized in other plants and organs/tissues (Shen et al, 2010; Pouvreau et al, 2011; Dussert et al, 2013; Ma et al, 2013) and the role of WRI1 has never been reported in palm endosperm.…”

MYB89 Transcription Factor Represses Seed Oil Accumulation

“…Previous studies indicated that WRI1 acts as the master regulator of FA biosynthesis by directly promoting the expression of SUS2, PI-PKb1, BCCP1, BCCP2, KASI, GLB1, and ROD1 and indirectly inducing ACP1, CAC2, CAC3, BIO2, PDH E1a, KASIII, MOD1, and LAS during Arabidopsis seed development (Focks and Benning, 1998;Cernac and Benning, 2004;Masaki et al, 2005;Baud et al, 2007;Maeo et al, 2009;To et al, 2012). ROD1 also was demonstrated to be up-regulated by overexpressing Arabidopsis WRI1 in N. benthamiana leaves (Grimberg et al, 2015) and castor (Ricinus communis) seeds (Adhikari et al, 2016). Thus, alterations in the FA profile of myb89 seeds become complicated and cannot be attributed to specific genes.…”

“…WRI1, which is the TF of the APETALA2-ethylene-responsive element-binding protein family, directly or indirectly targets some enzymes involved in the late glycolysis and plastidial fatty acid (FA) biosynthetic network (Cernac and Benning, 2004;Baud et al, 2007;Maeo et al, 2009). Mutations in wri1 cause an 80% reduction in Arabidopsis seed oil content (Focks and Benning, 1998), and overexpression of WRI1 leads to a significant increase in the oil content (Cernac and Benning, 2004;Maeo et al, 2009;Sanjaya et al, 2011;Grimberg et al, 2015;Adhikari et al, 2016). LEC1 encodes a protein that is homologous to the Saccharomyces cerevisiae HEME ACTIVATOR PROTEIN3 or mammalian NUCLEAR FACTOR YB subunit of the heterotrimeric CCAAT boxbinding factor (Lotan et al, 1998;Lee et al, 2003).…”

The effect of light conditions on interpreting oil composition engineering in Arabidopsis seeds

“…The seed oil content of HFA producing lines can be at least partially recovered by upregulating acetyl-CoA carboxylase (Adhikari, Bates, & Browse, 2016) or by coexpression of the hydroxylase with HFA selective TAG synthesis enzymes (RcDGAT2 or (van Erp et al, 2011;Bates et al, 2014;van Erp et al, 2015)). The more efficient incorporation of HFA into TAG by HFA selective DGAT or PDAT apparently reduces the negative effect of HFA on lipid metabolism indicated by a recovered in vivo acetyl-CoA carboxylase activity and more seed oil (Bates et al, 2014).…”

“…For example, variations in HFA content and oil quantity in the same genetic lines have been reported between different sets of experiments performed over a 10-year span (Adhikari et al, 2016;Bates et al, 2014;Bayon, Chen, Weselake, & Browse, 2015;Burgal et al, 2008;Lu et al, 2006;Lunn et al, 2018;van Erp et al, 2011van Erp et al, , 2015.…”