In many higher plants, seed oil accumulation is governed by complex multilevel regulatory networks including transcriptional regulation, which primarily affects fatty acid biosynthesis. Tree peony (Paeonia rockii), a perennial deciduous shrub endemic to China is notable for its seed oil that is abundant in unsaturated fatty acids. We discovered that a tree peony trihelix transcription factor, PrASIL1, localized in the nucleus, is expressed predominantly in developing seeds during maturation. Ectopic overexpression of PrASIL1 in Nicotiana benthamiana leaf tissue and Arabidopsis thaliana seeds significantly reduced total fatty acids and altered the fatty acid composition. These changes were in turn associated with the decreased expression of multitudinous genes involved in plastidial fatty acid synthesis and oil accumulation. Thus, we inferred that PrASIL1 is a critical transcription factor that represses oil accumulation by down-regulating numerous key genes during seed oil biosynthesis. In contrary, up-regulation of oil biosynthesis genes and a significant increase in total lipids and several major fatty acids were observed in PrASIL1-silenced tree peony leaves. Together, these results provide insights into the role of trihelix transcription factor PrASIL1 in controlling seed oil accumulation. PrASIL1 can be targeted potentially for oil enhancement in tree peony and other crops through gene manipulation.

WRINKLED1 (WRI1), a member of the APETALA2 (AP2) class of transcription factors regulates fatty acid biosynthesis and triacylglycerol (TAG) accumulation in plants. Among the four known Arabidopsis WRI1 paralogs, only WRI2 was unable to complement and restore fatty acid content in wri1-1 mutant seeds. Avocado (Persea americana) mesocarp, which accumulates 60-70% dry weight oil content, showed high expression levels for orthologs of WRI2, along with WRI1 and WRI3, during fruit development. While the role of WRI1 as a master regulator of oil biosynthesis is well-established, the function of WRI1 paralogs is poorly understood. Comprehensive and comparative in silico analyses of WRI1 paralogs from avocado (a basal angiosperm) with higher angiosperms Arabidopsis (dicot), maize (monocot) revealed distinct features. Predictive structural analyses of the WRI orthologs from these three species revealed the presence of AP2 domains and other highly conserved features, such as intrinsically disordered regions associated with predicted PEST motifs and phosphorylation sites. Additionally, avocado WRI proteins also contained distinct features that were absent in the nonfunctional Arabidopsis ortholog AtWRI2. Through transient expression assays, we demonstrated that both avocado WRI1 and WRI2 are functional and drive TAG accumulation in Nicotiana benthamiana leaves. We predict that the unique features and activities of ancestral PaWRI2 were likely lost in orthologous genes such as AtWRI2 during evolution and speciation, leading to at least partial loss of function in some higher eudicots. This study provides us with new targets to enhance oil biosynthesis in plants.

In higher plants, acyl-CoA:diacylglycerol acyltransferase (DGAT) and phospholipid:diacylglycerol acyltransferase (PDAT) catalyze the terminal step of triacylglycerol (TAG) synthesis in acyl-CoA-dependent and -independent pathways, respectively. Avocado (Persea americana) mesocarp, a nonseed tissue, accumulates significant amounts of TAG (~70% by dry weight) that is rich in heart-healthy oleic acid (18:1). The oil accumulation stages of avocado mesocarp development coincide with high expression levels for type-1 DGAT (DGAT1) and PDAT1, although type-2 DGAT (DGAT2) expression remains low. The strong preference for oleic acid demonstrated by the avocado mesocarp TAG biosynthetic machinery represents lucrative biotechnological opportunities, yet functional characterization of these three acyltransferases has not been explored to date. We expressed avocado PaDGAT1, PaDGAT2, and PaPDAT1 in bakers’ yeast and leaves of Nicotiana benthamiana. PaDGAT1 complemented the TAG biosynthesis deficiency in the quadruple mutant yeast strain H1246, and substantially elevated total cellular lipid content. In vitro enzyme assays showed that PaDGAT1 prefers oleic acid compared to palmitic acid (16:0). Both PaDGAT1 and PaPDAT1 increased the lipid content and elevated oleic acid levels when expressed independently or together, transiently in N. benthamiana leaves. These results indicate that PaDGAT1 and PaPDAT1 prefer oleate-containing substrates, and their coordinated expression likely contributes to sustained TAG synthesis that is enriched in oleic acid. This study establishes a knowledge base for future metabolic engineering studies focused on exploitation of the biochemical properties of PaDGAT1 and PaPDAT1.

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Gluconeogenesis (GNG) is the process of regenerating glucose and NAD+ that allows for continued ATP synthesis by glycolysis during fasting or in hypoxia. Recent data from C. elegans and crustaceans challenged with hypoxia show differential and tissue-specific expression of GNG-specific genes. Here we report differential expression of several GNG-specific genes in the head and body of a model organism, Daphnia magna, a planktonic crustacean, in normoxic and acute hypoxic conditions. We predict that GNG-specific transcripts will be enriched in the body, where most of the fat tissue is located, rather than in the head, where the tissues critical for survival in hypoxia, the central nervous system and locomotory muscles, are located. We measured the relative expression of GNG-specific transcripts in each body part by qRT-PCR and normalized them by either the expression of a reference gene or the rate-limiting glycolysis enzyme pyruvate kinase (PK). Our data show that of the three GNG-specific transcripts tested, pyruvate carboxylase (PC) showed no differential expression in either the head or body. Phosphoenolpyruvate carboxykinase (PEPCK-C), on the other hand, is upregulated in hypoxia in both body parts. Fructose-1,6-bisphosphatase (FBP) is upregulated in the body relative to the head and upregulated in hypoxia relative to normoxia, with a stronger body effect in hypoxia when normalized by PK expression. These results support our hypothesis that Daphnia can survive hypoxic conditions by implementing the Cori cycle, where body tissues supply glucose and NAD+ to the brain and muscles, enabling them to continuously generate ATP by glycolysis.